Background: Maximum safe resection of infiltrative brain tumors in eloquent area is the primary objective in surgical neuro-oncology. This goal can be achieved with direct electrical stimulation (DES) to perform a functional mapping of the brain in patients awake intraoperatively. When awake surgery is not possible, we propose a pipeline procedure that combines advanced techniques aiming at performing a dissection that respects the anatomo-functional connectivity of the peritumoral region. This procedure can benefit from intraoperative monitoring with computerized tomography scan (iCT-scan) and brain shift correction. Associated with this intraoperative monitoring, the additional value of preoperative investigation combining brain mapping by navigated transcranial magnetic stimulation (nTMS) with various neuroimaging modalities  has not yet been reported.Case Report: A 42-year-old left-handed man had increased intracranial pressure (IICP), left hand muscle deficit, and dysarthria, related to an infiltrative tumor of the right frontal lobe with large mass effect and circumscribed contrast enhancement in motor and premotor cortical areas. Spectroscopy profile and intratumoral calcifications on CT-scan suggested an WHO grade III glioma, later confirmed by histology. The aforementioned surgical procedure was considered, since standard awake surgery was not appropriate for this patient. In preoperative time, nTMS mapping of motor function  was performed, combined with magnetic resonance imaging (MRI)-based tractography reconstruction of 6 neural tracts  and resting-state functional MRI connectivity (rs-fMRI) of sensorimotor and language networks. In intraoperative time, DES mapping was performed with motor evoked response recording and tumor resection was optimized using non-rigid image transformation of the preoperative data  to iCT data. Image guidance was updated with correction for brain shift and tissue deformation using biomechanical modeling taking into account brain elastic properties. This correction was done at crucial surgical steps, i.e., when tumor bulged through the craniotomy after dura mater opening and when approaching the presumed eloquent brain regions. This procedure allowed a total resection of the tumor region with contrast enhancement as well as a complete regression of IICP and dysarthria. Hand paresis remained stable with no additional deficit. Postoperative nTMS mapping confirmed the good functional outcome.Conclusion: This case report and technical note highlights the value of preoperative functional evaluation by nTMS updated intraoperatively with correction of brain deformation by iCT. This multimodal approach may become the optimized technique of reference for patients with brain tumors in eloquent areas that are unsuitable for awake brain surgery. Maximum safe resection of infiltrative brain tumors in the eloquent areas is the primary objective in surgical neuro-oncology. Intraoperative direct electrical stimulation (DES) is the standard of care to map motor cortex function . The functional location of the primary motor cortex (M1) as defined by nTMS was used as region of interest (ROI) for tractography reconstruction, in place of the standard anatomical location of M1, which is severely distorted as a result of tumor infiltration or perilesional edema. On the other hand, the five main speech tracts were reconstructed using ROI-based tractography by an experienced neuroradiologist.In addition, functional brain connectivity of rs-networks was appraised by rs-fMRI. This approach is especially helpful to identify functional brain networks in patients with neurological impairments who would be unable to comply with complex tasks.3. Intraoperative iCT-scan with BSCThis correction was performed at crucial steps, i.e., after opening the dura and halfway during tumor resection.The first step was to implement all the aforementioned data  in the iCT before craniotomy in the OR.A second step was to acquire iCT during surgical resection when a significant brain shift was likely to have occurred.Then, elastic fusion of the iCT to the preoperative brain MRI was performed for BSC.Preoperative nTMS as well as motor nTMS-guided tractography, speech anatomical tractography and motor and speech rs-fMRI data were updated in the virtual MRI with BSC. This procedure allowed the neurosurgeon to keep performing a high-resolution morphological image-guided surgery through virtual MRI.Finally, nDES was performed cortically and subcortically to optimize the extent of the resection with minimization of motor function deficit.The application of this pipeline in the timeline of care for the reported case is displayed in A 42-year-old left-handed man presented to the emergency unit of our hospital  with left hand paresis, left upper limb partial motor seizures and headaches that started 1 month earlier. The patient had a history of epilepsy diagnosed when he was 32, followed by his general practitioner without further neuroimaging exploration or antiepileptic drug prescription. Clinically, the neurosurgeon on call found left hand muscle deficit (3/5), dysarthria, and an IICP syndrome with clouding of consciousness, headache, nausea and vomiting.An urgent brain MRI showed a 6 cm long infiltrative tumor of the right frontal lobe with tissular and kystico-necrotic portions, associated with large mass effect and circumscribed contrast enhancement in motor and premotor cortical areas.The spectroscopy profile and intratumoral calcifications on CT-scan suggested an WHO grade III glioma . During Prior to surgery, nTMS mapping of motor function was performed using Navigated Brain Stimulation (NBS) system . The 3D T1-weighted brain MRI was co-registered to the patient's head using anatomical landmarks and surface matching. EMG surface electrodes  were placed on deltoid, first dorsal interosseous (FDI), and tibialis anterior (TA) muscles of the right side with an additional large adhesive ground electrode placed over the forearm. Cortical mapping was performed according to nTMS Workshop Report  was represented at the normal-appearing posteromedial edge of the tumor in the posterior part of the precentral cortex. TA-CM was also equally represented in the T1 hypointensity parasagittal cortex (superior to the FDI-CM) and in the normal-appearing posterior part of the superior frontal gyrus.3, 190 slices.MR imaging was performed on a 3T Verio  imager with a 32-channel head coil. Structural image was acquired using a 3D magnetization-prepared, rapid acquisition gradient echo (MP-RAGE) sequence with the following parameters: TR: 2,400 ms, TE: 3.65 ms, TI: 1,000 ms, flip angle: 8°, voxel size: 1 × 1 × 1 mm2/s. For speech tracts , and acceleration factor: 2. Diffusion weighting was encoded along 12 independent orientations using a b-value of 1,000 mmh tracts , we usedh tracts  using boh tracts  using thh tracts   and statistical parametric mapping SPM12 software  were used for image pre-processing.Whole brain rs-fMRI data were acquired using a T2The processing included slice-timing correction, head motion correction, gray/white matter/cerebrospinal fluid structural segmentation. EPI images were coregistered to anatomical images and a spatial smoothing using a Gaussian filter with a full width at half maximum of 8 mm was then performed. Seed-based correlation analyses were performed using the CONN toolbox, which is an open-source Matlab/SPM-based cross-platform software for the analysis of functional connectivity  and the BSC was performed with elastic fusion using the Brainlab virtual MRI software (BrainLAB) with biomechanical simulation based on a finite element model taking into account the elastic properties of various brain regions  and a Nimbus stimulator  combined with Brainlab neuronavigation tracking device  and keeping a bispectral index superior to 55. The DES settings were as usual   and within 3 weeks it improved compared to the preoperative status (4/5). There was no additional deficit in the immediate postoperative time.A post-operative nTMS mapping performed 3 weeks after the surgery confirmed the correct functional outcome, with a reorganization of the CM of the three muscles evaluated preoperatively . CompareThis technical report underlines the value of preoperative functional and structural evaluation combining nTMS and various neuroimaging modalities (tractography and rs-fMRI) subsequently updated intraoperatively with BSC and elastic fusion between iCT and preoperative MRI. This multimodal approach may become the optimized technique of reference for patients with brain tumors in eloquent areas who do not qualify for awake brain surgery  predictive value is well-established in the literature  or magnetoencephalography (MEG) with source localization of sensory-evoked potentials or magnetic fields to map the sensorimotor cortex appears promising  (Vitikainen et al., In conclusion, the combination of preoperative mapping advanced techniques, asleep intraoperative motor nDES, and iCT with BSC and elastic fusion to preoperative brain MRI in a patient not eligible for awake craniotomy allowed for complete resection of tumor portions with contrast enhancement without permanent neurological deficits. Here, preoperative nTMS was only used to perform motor mapping, but our proposed pipeline could benefit in the future from preoperative nTMS mapping for speech, visual and cognitive functions.In our case report, the patient felt reassured to be operated following the M-iCT BSC procedure, which can be considered an “asleep awake-surgery” procedure. Although he did not benefit from AC, the patient could still feel involved in every step of the surgical treatment through our procedure. First, he could contribute to his surgery in optimal conditions: lying in a comfortable chair, skull closed, with the possibility to take some rest during the mapping session. Second, he could understand the exact surgical strategy, the anatomical, tractography and functional reconstructions being shown to him on a screen before surgery. He was fully aware of the risks and thus able to accept or refuse them. Third, on the basis of the postoperative nTMS mapping procedure, he better understood how to perform efficient neurorehabilitation to improve motor functions and also why a subsequent resection surgery might be eligible.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.Written informed consent was obtained from the individual for the publication of any potentially identifiable images or data included in this article.SS, BBar, and J-PL were responsible for the initial conception and draft of this manuscript. SS and BBar collected and analyzed data and prepared revised drafts and figures. BBar and SA carried out navigated TMS investigation. PB and BBap carried out neuroimaging investigation. SS, ST, KA, OL, KE, and SP were involved or responsible concerning the work-up of the patient, planning and conducting surgery, and providing clinical care. All authors contributed to the article and approved the submitted version.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.


Chromatin architecture is highly dynamic during different phases of cell cycle to accommodate DNA-based processes. This is particularly obvious during mitotic exit, where highly condensed rod-like chromatids need to be rapidly decondensed. Such chromatin structural transitions are tightly controlled and organized as any perturbance in this dynamic process can lead to genome dysfunction which may culminate in loss of cellular fitness. However, the mechanisms underlying cell cycle-dependent chromatin structural changes are not fully understood. In this mini review, we highlight our current knowledge of chromatin structural organization, focusing on mitotic exit. In this regard, we examine how nuclear processes are orchestrated during chromatin unfolding and compartmentalization and discuss the critical importance of cell cycle-controlled chromatin landscaping in maintaining genome integrity. The cell cycle of proliferating cells is defined by two major events, first, error-free duplication of the genome during synthesis phase and second, faithful transmission of genetic material into the daughter cells during mitosis. Since genetic material is packaged in the form of chromatin, the proper execution of nuclear processes is critically dependent on cell cycle regulated chromatin organization and restructuring . This prThe fundamental repeating unit of chromatin is the nucleosome, which is formed by ~147 bp of DNA wrapped around an octamer histone core . IndividHigh-throughput sequencing based approaches have markedly advanced the understanding of chromatin folding patterns and their relevance to nuclear functions. In particular, different versions of chromosome conformation capture-based methods  were devBox 1Condensed chromatin regions corresponding to B-type domains that lie in proximity to the nuclear lamina (NL) are termed as lamina-associated domains (LADs) . There aNucleolar-associated domains (NADs)Nucleolar-associated domains (NADs) are heterochromatic regions that associate with the nucleolus . NADs arThe massive structural reorganization of chromatin during mitosis is mediated by the eukaryotic members of SMC  protein complexes, namely condensins and cohesins . TogetheIn addition to histone H3 phosphorylation, different methylation states of histone H4 lysine 20 (H4K20me1/2/3) have also been implicated in chromatin compaction . HoweverMitotic exit is characterized by two major nuclear events, first, the nuclear envelope is reformed to provide an enclosed space for the segregated genomic material. Second, the re-establishment of functional interphase chromatin within the nuclear envelope, where rod-shaped chromatids rapidly decondense into more loosely arranged, non-random structures, fully competent for DNA-based processes. Indeed, simulation on a mitotic chromosome-like polymer shows that the large-scale 3D organization of TADs and A/B compartments during mitotic exit occurs simply as a result of partial decondensation in an inflation-like process . In thisIn the context of this review, we focus on how the major B-type compartment components i.e. LADs and NADs, are organized at mitotic exit. During interphase, LADs interact dynamically with the NL however, they move only within a layer <1 µm thick . FurtherNADs are also found to locate in the proximity of NL in a subset of cells. In this regard, there may be a substantial overlap between NADs and LADs with some studies showing that these loci could switch positions following mitotic exit . A recenAt the nucleosomal level, the chromatin landscaping at mitotic exit is marked primarily by reappearance of histone acetyl marks and loss of histone phosphorylation . The phoAdditional protein complexes have also been shown to play a role in chromatin decondensation at the mitotic exit. Firstly, removal of the mitotic kinase Aurora B from the chromatin seems to be a prerequisite for chromatin decondensation and nuclear envelope reformation. This is carried out by the hexameric ATPase p97 that binds to the ubiquitylated form of Aurora B and evicts it from the chromatin thereby, facilitating chromatin decondensation . Apart fEstablishing proper ‘ground state’ chromatin structure entails massive structural reorganization of the chromatin. Using single-cell Hi-C analysis, Nagano et al. compared chromatin structure in different cell cycle phases, starting from mitotic exit . As the de novo establishment of functional chromatin domains needs to be well coordinated with the genome-wide execution of DNA-based processes in particular transcription and replication. In this regard, it is unclear at the moment whether nuclear functions  drive chromatin domain unfolding or vice versa. Below we discuss how cells coordinate chromatin reorganization and nuclear processes during their transition to the next cell cycle.The large-scale spatial segregation of locally folded loops, TADs, compartments that define interphase 3D chromatin organization is largely absent in mitotic chromosomes . Thus, dTo achieve maximum chromatin condensation during mitosis, the landscape of interphase chromatin including intra- and inter-chromosomal contacts is lost. In this regard, many chromatin modifiers and transcription factors are dissociated from chromatin, facilitating segregation of genomic material into the daughter nuclei . In contAdditionally, recent evidence indicates ongoing transcription of many genes during mitosis albeit at low levels, with a transient surge at the mitotic exit . The iniEstablishing adequate chromatin compaction during G1 phase is also necessary for preventing unregulated transcription. Using DNMT (DNA methyltransferase) and HDAC (histone deacetylase) inhibitors, Brocks et al. showed that disruption of repressive chromatin environment induces cryptic transcription start sites encoded within long terminal repeat retrotransposons . Recent DNA replication is a tightly controlled chromatin process that ensures faithful duplication of genetic material once per cell cycle. Replication is temporally divided into two-steps, where first the future replication origins are ‘licensed’ by loading of pre-RC (pre-replication complex) starting in late telophase and continued through G1 phase, followed by ‘firing’ of origins at the start of S phase . Pre-RC Chromatin enforces specificity of replication initiation by restricting non-specific ORC binding to origins . Thus, tde novo establishment of chromatin state (cLADs vs fLADs) at mitotic exit. It is unclear how cells push certain LAD regions to the nuclear periphery while others remain in the nuclear interior. The question remains if chromatin landscaping at mitotic exit is largely a stochastic process or there is a method to this randomness. By employing high throughput ‘omics’ approaches, future studies will shed light on chromatin landscaping at mitotic exit and how it regulates nuclear processes thereby maintaining genome integrity and cell identity.To coordinate and regulate various nuclear functions, chromatin inherited by the daughter cells during mitotic exit maintains the structural organization of their predecessor. It is an important mechanism for cells to maintain their cellular identity. This inevitably requires highly regulated chromatin decondensation, which is dictated by both chromatin modifications and non-histone chromatin structural proteins. The molecular events leading to reversal of highly condensed chromosomes into loosely organized interphase chromatin are not fully elucidated. In this regard, several key questions require further investigations. A comprehensive analysis of chromatin factors that remain on mitotic chromosomes during cell division is lacking. Also, it is unresolved at the moment how much mitotic bookmarking contributes to reestablishment of interphase chromatin states and how extensive it is throughout the genome. For certain genomic regions such as LADs, there is a MS, NN, and CS contributed to the conception, writing, and checking of the manuscript for important intellectual content.This work was created by MS, NN, and CS based on the support from the Benzon Foundation, The Novo Nordisk Foundation, The Danish Cancer Society, and The Danish Medical Research Council.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.


Gastrointestinal (GI) cancers are among the most common and lethal solid tumors worldwide. Unlike in malignancies such as lung, renal and skin cancers, the activity of immunotherapeutic agents in GI cancers has, on the whole, been much less remarkable and do not apply to the majority. Furthermore, while incremental progress has been made and approvals for use of immune checkpoint inhibitors (ICIs) in specific subsets of patients with GI cancers are coming through, in a population of ‘all-comers’, it is frequently unclear as to who may benefit most due to the relative lack of reliable predictive biomarkers. For most patients with newly diagnosed advanced or metastatic GI cancer, the mainstay of treatment still involves chemotherapy and/or a targeted agent however, beyond the second-line this paradigm confers minimal patient benefit. Thus, current research efforts are concentrating on broadening the applicability of ICIs in GI cancers by combining them with agents designed to beneficially remodel the tumor microenvironment (TME) for more effective anti-cancer immunity with intention of improving patient outcomes. This review will discuss the currently approved ICIs available for the treatment of GI cancers, the strategies underway focusing on combining ICIs with agents that target the TME and touch on recent progress toward identification of predictors of sensitivity to immune checkpoint blockade in GI cancers. Gastrointestinal cancers are a huge global health problem. In 2018 almost 5 million new cases were diagnosed [Over the past decade immunotherapy has revolutionized cancer treatment. Immune checkpoint inhibitors (ICIs) are now the cornerstone of managing malignancies such as lung, renal and skin cancers, among others. In some cases patients are experiencing marked radiological responses which may be sustained for years and side effects can be minimal compared with traditional chemotherapies. For an increasing number, this has meant a better quality and longer life. However, the activity of immunotherapeutic agents in GI cancers has, on the whole, been much less striking. Furthermore, in a population of ‘all-comers’, it is frequently unknown who may benefit due to the relative lack of reliable predictive biomarkers. For the majority of patients with newly diagnosed advanced/metastatic GI cancer, the mainstay of treatment still involves chemotherapy and/or a targeted agent. However, chemotherapy and/or targeted agents beyond the second-line have minimal efficacy in these diseases. More efficacious treatments are urgently needed.ICIs work by inhibiting the interaction between an immunosuppressive ligand or signal and their corresponding receptor or protein on immune or tumor cells, thus restoring the pre-existing host immune response against cancer . ExampleThis article will discuss the currently approved ICIs available for the treatment of GI cancers, strategies underway focusing on combining ICIs with agents that target the TME and touch on recent progress toward predictive biomarker identification. Combinations of ICIs with cancer vaccines, cellular therapies or cytokine based therapies will not be covered.The cancer-immunity cycle, a term first coined by Chen and Mellman in 2013, describes the seven main steps required for the immune system to carry out its final effector function of killing of cancer cells . The majIn 2017, Chen and Mellman went on to describe three distinct immune phenotypes which could explain the mechanism of a tumour’s resistance to anti-cancer immunity .Resistance to ICIs may be present from the outset or develop over a period of time on treatment due to mechanisms either intrinsic or extrinsic to the tumor cell , 10. OveDetailed insight into TME components and their role in de novo resistance to checkpoint blockade in GI cancers has recently been reviewed by Batista et al. , and broIn 2017, the FDA approved both pembrolizumab and nivolumab, for use in the second-line treatment of MSI-H or dMMR metastatic colorectal cancer (mCRC) following the results of two pivotal trials. These studies demonstrated objective response rates (ORR) of 40%  in patieIn 2017 the FDA also granted the first tumor-agnostic approval to pembrolizumab for the treatment of any dMMR or MSI-H tumor following progression after standard treatment. The KEYNOTE-158 trial, which assessed the efficacy of 3 weekly pembrolizumab in 233 patients with 27 different types of advanced or metastatic non-colorectal MSI-H/dMMR tumors, subsequently reported an ORR of 33.4% in all patients which was durable . This daThe role of ICIs in esophagogastric (OG) cancers is complex and rapidly evolving, with ongoing questions as to the most appropriate timing of immunotherapy. However, there now is established evidence for ICI use, particularly PD-1 inhibitors, in these tumors Table .KEYNOTE 181 was a phase III study in patients with advanced GOJ or esophageal cancer, showing superiority of pembrolizumab over investigator choice chemotherapy, producing prolonged median overall survival (mOS) of 9.3 vs. 6.7 months in 222 patients with PD-L1 positive tumors (CPS ≥ 10) . PatientTwo phase Ib studies (KEYNOTE 12 and KEYNOTE 28) have evaluated the role of pembrolizumab in PD-L1 expressing gastric cancer and esophageal cancer (SCC and adenocarcinoma), respectively , 30. BotNivolumab has efficacy in advanced gastric and GOJ adenocarcinoma in the third line setting regardless of PD-L1 expression, as seen in the ATTRACTION-2 study . In thisTrials of single-agent immune checkpoint inhibitors have been particularly disappointing in pancreatic cancer. In a phase II study of ipilimumab monotherapy in 27 patients with advanced pancreatic ductal adenocarcinoma (PDAC), one patient had an objective response after initial pseudoprogression however there were no responses in the other 26 patients  SimilarlFollowing favorable results from the CheckMate 040 study, the FDA granted accelerated approval to nivolumab in September 2017 (Table Biliary tract cancers (BTCs) are rare malignancies so data from trials of checkpoint blockade in patients with BTC have come from basket trials with BTC cohorts. In KEYNOTE-028, pembrolizumab was given to patients with PD-L1 positive tumors including 24 patients with heavily pretreated PD-L1 positive BTC . The ORRHuman papilloma virus (HPV) infection has been associated with higher numbers of tumor infiltrating lymphocytes and up-regulation of PD-1 checkpoints and is attributable to over 80% of case of squamous cell carcinoma of the anal canal (SCCA) . In the n = 12, 54 patients in total in the study) and patients with TMB < 12 mutations/Mb were defined as TMB-Low [As more and more approvals are coming through for use of ICIs in patients with GI cancers, the key research focus has been to try and improve and refine patient selection to expand the potential benefit of these therapies. In parallel to MSI-H/dMMR status discussed above, tumor TMB status, a reflection of the number of mutations within the tumor and thus the presumed neoantigen load capable of triggering an immune response , is an i TMB-Low . TMB-Hig TMB-Low . In anot TMB-Low . It does TMB-Low  and so,  TMB-Low . Blood-bPD-L1 expression is another important biomarker to mention, not least because a number of approved uses of ICIs in esophagogastric cancers mandate tumor PD-L1 positivity Table . HistoriVirus-associated cancers also demonstrate increased mutational loads , 60 and While the candidate predictive biomarkers so far discussed have been helpful in determining which patients with GI cancers may benefit most from ICI therapy, more precise biomarkers are clearly needed. Another potential contender on the horizon is the gut microbiome particularly in colorectal cancer where, for example, a certain species of bacterium has been shown to be inversely correlated to levels of T cell infiltrates in MSH-H CRC  and thusTraditional cytotoxic chemotherapy exerts its anti-neoplastic activity through direct cytotoxicity, impacting the cell cycle and leading to apoptosis. However, studies have shown that some chemotherapeutic agents also have immune modulating effects, through down-regulating immune inhibitory cells and stimulating the production of pro-inflammatory cytokines, leading to a ‘hot’ or T cell inflamed TME  Table 33. Combinn = 25) determined that pembrolizumab combined with platinum/fluropyrimidine in the first-line treatment of advanced gastric cancer had a manageable safety profile and, furthermore, showed impressive ORR (60%) and mOS of 13.8 months [The KEYNOTE 059 trial  regulates the tumoral vascular environment which enhances angiogenesis , and is There is evidence that VEGF also has immune-modulating effects, through impacting DCs and inhibiting T cell activity  on tumor infiltrating lymphocytes (TILs). Blockade of this inhibitory signal can promote T cell mediated tumor cell death and activate TILs in CRC typically express high levels of LAG3, which make this a promising therapeutic target . FurtherEpigenetics is the modification of gene expression without alteration of DNA’s nucleic acid sequence . These mVarious epigenetic therapies have been approved for use in the treatment of hematological malignancies however their use as single agents in solid tumors, including CRC, has been historically somewhat limited for reasons such as short half-lives, high toxicity or low or no responses . This haBy far the most studied epigenetic modulators are inhibitors of histone deacetylases (HDACi) and DNA methyltransferases (DNMTi). Pre-clinical testing demonstrates that the DNMTi 5-azacitadine (5-AZA) upregulated 15 gene sets involved in the immune response such as antigen presentation, interferon signaling and chemokine and cytokine signaling in colorectal cancer cell lines . Mouse mSeveral clinical studies of checkpoint inhibitors combined with epigenetic modifiers, including IDO inhibitors which have also demonstrated similarly favorable immunomodulatory effects pre-clinically , are curAbberations in the Wnt/β-catenin signaling pathway occur frequently in cancer and while the constituents of the pathway are known, the mechanisms underpinning how the proteins interact are yet to be fully elucidated . The rolPreliminary clinical studies of Wnt signaling modulators as monotherapies including Porcupine inhibitors, Frizzled receptor targeting agents, a Wnt5a-mimetic and agents targeting components further downstream in the pathway, have so far demonstrated promising safety profiles in patients with GI cancers where side effects including bone toxicity appear to be manageable –116, howFirstly, activated Wnt/β-catenin signaling appears to accentuate tumor immune exclusion by suppressing DC recruitment into the TME via down-regulation of the chemokine CCL4 as well as impaired priming of effector T cells in melanoma mouse models, which were subsequently resistant to immune checkpoint blockade , 119. SuIn the clinic, the combination of DKN-01 and pembrolizumab has been shown to be safe and tolerable in patients with advanced MSS OG cancer with a hint of potential efficacy as a PR was observed in 1 patient and 5 patients had SD . InteresThe tumor stroma is comprised of dense connective tissue including CAFs, stromal cells, osteoblasts, chondrocytes and ECM and it is a critical component of the TME . ActivatHigh levels of focal adhesion kinases (FAK) in PDAC appear to correlate with low levels of CTLs and a generally immunosuppressive TME and in mouse models FAK inhibition (FAKi) appeared to reduce tumor fibrosis and halt PDAC progression as well as decrease populations of immunosuppressive cells such as TAMs and Tregs . FAKi alAgents targeting chemokine proteins and chemokine receptors which are involved in cell migration have also demonstrated potential as valuable ICI partners in PDAC. For example, the chemokine CXCL12 secreted by CAFs and the chemokine receptor CXCR4 found on T cells appear to drive immunosuppression in the TME by increasing populations of MDSCs , 133. CXAlternative combination approaches include use of Bruton’s tyrosine kinase (BTK) inhibitors, which have been shown to deplete MDSCs in the TME, improve CTL activity and ameliorate the fibrous stroma of PDAC pre-clinically –141 withOver the past few years, data supporting clinical benefit for immune checkpoint inhibitors in the treatment of gastrointestinal cancers has gradually mounted and approvals for specific indications have come through. However, the challenge of expanding the benefit of immunotherapy to the majority of the population with non-T cell inflamed, ‘cold’ gastrointestinal tumors with intrinsic resistance to these therapies is still very much an unmet need. Research efforts into effective predictive biomarker identification, fraught with hurdles such as intratumoral heterogeneity, is still being explored and refined however much headway has already been made. Furthermore, rational combinatorial strategies of immune checkpoint inhibitors with agents that produce beneficial immunomodulatory effects and reprogram the TME to overcome intrinsic resistance to effective anti-cancer immunity, hold significant promise. The corresponding translational components of the clinical studies discussed herein may be the key to gaining a deeper understanding of underlying mechanisms of response and resistance to these strategies and slowly unlock more about the intricacies of the immune milieu in these malignancies with the hope of discovering more efficacious treatments for our patients.


Inflammatory bowel disease (IBD), comprising ulcerative colitis and Crohn’s disease, is characterized by widespread inflammation of the gastrointestinal tract with systemic manifestations. Inflammation is one of the driving forces for the pathogenesis of atherosclerosis and its dreaded complications like myocardial infarction (MI). Yet, the association between IBD and myocardial infarction has not been thoroughly established.Myocardial infarction in IBD patients was predominantly seen in young women during the active disease process. At the same time, elevated levels of C-reactive protein and other pro-inflammatory markers were observed in both IBD and atherosclerosis. Increasing evidence suggests inflammation inhibits fibrinolysis, expresses procoagulants, and suppresses anticoagulants promoting thrombosis formation. Moreover, the alteration of gut microbiota impacts the pathogenesis of inflammation and predisposes one to ischemic heart disease.Accordingly, all IBD patients should be screened and counseled on lifestyle modifications for the traditional risk factors of atherosclerosis. Future researchers should consider conducting more clinical trials on anti-inflammatory medication targeting atherosclerosis and therapeutics, while targeting the gut microbiota to reverse the inflammatory atherosclerotic process. Inflammatory bowel disease (IBD) is characterized by widespread chronic inflammation of the gastrointestinal tract. The two central pathologies that compose IBD include ulcerative colitis and Crohn’s disease. Patients are typically diagnosed between the ages of 15-35 years, routinely present with abdominal pain, diarrhea, bloating, rectal bleeding, abdominal cramps, and weight loss . The disInflammatory bowel disease is considered idiopathic in nature, but factors that predispose the condition include immune response, genetic, environmental, and microbial factors . The chrAt the same time, studies have shown that inflammation plays a critical role in the development of atherosclerosis. This process is regulated by an interaction between pro-atherogenic inflammatory and anti-inflammatory mediators, which can lead to the development of thrombosis, and increase susceptibility to coronary artery disease (CAD). Proatherogenic inflammatory mediators consist of T-helper cell type 1 and lymphocyte B2 cells, while regulatory T-cells and types of T-helper 2 related cytokines illustrated an anti-atherogenic effect. Long-standing elevation of levels of C-reactive protein, interleukin 6 (IL-6), activated protein C is now widely considered independent risk factors for coronary artery disease .Many experts widely document that chronic inflammation increased the incidence of cardiovascular manifestations. But, the association between IBD and myocardial infarction (MI) has not been entirely established. ,9.Recent studies have illustrated mixed results: Some showed an association , while oIt is essential to establish the association between the two conditions so that measures can be taken to start preventing the disease. The purpose of this review is to examine the current literature regarding inflammatory bowel disease and myocardial infarction and establish the relationship between the two conditions.Although it predominantly presents in Western societies, cases of inflammatory bowel disease have risen worldwide. Ulcerative colitis and Crohn’s disease are grouped in the same disease process; however, subtle differences exist between the two inflammatory disorders, as can be seen in Table According to a study assessing the global and national burden of Inflammatory bowel disease, the prevalence of IBD in the United States of America was reported to range from 252 to 439 cases per 100,000 population. The evidence of IBD revealed to be more prevalent in females compared to males. Due to the chronic nature and low mortality of the disease it’s prevalence is high and more complications are encountered. .Genetic, environmental factors and microbial factors in IBD and cardiovascular diseaseAlthough inflammatory bowel disease (IBD) is widely accepted as a chronic inflammatory process, the exact cause is unknown. It is stated that there are interactions between a wide array of environmental, host, microbial, and genetic factors .Role of Genetic FactorsAnalyzing IBD patients’ genetic material, illustrated that there is a distinctive locus that regulates intracellular bacteria-killing, innate immunity, and adaptive immune responses. A lesion in that locus could impair the immune system, upregulate cytokines, and disrupt bacteria clearance contributing to the chronic inflammatory process .Role of Environmental FactorsEnvironmental factors play a significant role in the disease process. Studies confirm smoking has a protective effect in patients with ulcerative colitis, as these patients reporting lower rates of relapse, and hospitalizations; however, smoking showed a completely inverse association in patients with Chron’s disease. Cigarette smoking predisposed patients to a more complicated disease course despite aggressive therapy and an increased requirement for surgery. Hence, smoking cessation is considered one of the most critical lifestyle modifications recommended to Crohn’s disease patients. Although it is crucial to establish smoking has no therapeutic effect in patients with ulcerative colitis, patients with a prior habit should not be discouraged from quitting due to the benefits outweighing the risk. Other environmental factors studied include vitamin D deficiency and increased stress, which showed an increased susceptibility to develop inflammatory bowel disease. ,15-17. Also, body mass index (BMI) correlated with the active disease process in Chrons disease, as an increase in extraintestinal manifestations was observed in patients with high BMI, suggesting that chronic inflammation associated with obesity may play a role in the disease process. At the same time, obesity and smoking are two of the most notorious risk factors for myocardial infarction. Accordingly, they both contribute to the active disease process for Inflammatory bowel disease, insinuating an indirect association between the two-disease process.Role of the Gut MicrobiomeThe gut microbiome, which develops and stabilizes in the second week of life, has around 1150 bacterial organisms that have shown multiple pathological changes throughout the IBD process, termed as dysbiosis. The predominant bacteria in healthy adults arise from the phyla: Bacteroidetes and Firmicutes. These, along with Actinobacteria and Proteobacteria, comprise 99% of the intestinal bacteria. The gut microbiota provides many vital functions such as nutrition, metabolism, contributing to bacterial defense, and immune function. The bacteria regulate the development of various types of T-cells, like regulatory T-cells and Th17 cells. (T-helper 17 cells) Regulatory T-cells have shown anti-inflammatory properties by suppressing different types of immune cells in the immune system. T-helper 17 cells (Th17) cells are characterized by their ability to produce various interleukins (IL) such as IL-17A, IL-17F, IL-21, and IL-22 in the gut microbiota. A decrease in bacteria with anti-inflammatory properties has been the culprit in patients with IBD, evidence stemming from comparing the gut microbiota of an IBD patient to a healthy person -20.There was an abundance of Proteobacteria such as E. coli and Bacteriodetes reported, while anti-inflammatory organisms like Firmicutes are decreased. The surplus bacteria with adhering properties like E. coli contribute to alteration in the gut bacteria, instigating an immune response leading to extensive inflammation. Metabolites such as hydrogen-sulfate produced from bacteria damage the intestine’s epithelial cells disrupting the barrier function, which increases intestinal permeability and inducing more mucosal inflammation. When the barrier function is compromised, alterations in the tight epithelial junctions can enhance the activity of inflammatory cytokines such as tumour necrosis factor alpha , interferon-gamma, and interleukins IL-13 resulting in systemic endotoxemia -20.Studies show several disorders affecting the heart, such as obesity and metabolic syndrome linked to alteration or deficiency in the gut microbiota. An increased amount of Firmicutes species have been linked to an obese person compared to a lean person. Depending on the bacteria present, a number of patients have weight gain and elevated blood sugars compared to someone consuming the same number of calories .Obesity and metabolic syndrome predispose to atherosclerosis and coronary artery disease (CAD) representing another indirect association between inflammatory bowel disease and myocardial infarction. Hypercholesterolemia and hypertension are the two most important etiologies triggering MI; therefore, it’s important to consider screening for the specific markers that predict CAD and start prophylaxis to prolong or completely prevent MI.Consequences of inflammation in IBD activity on the cardiovascular system Pathophysiology of inflammation in IBD activity Dysfunction in the innate and adaptive immune responses have been well established in inflammatory bowel disease. The intestinal lamina propria houses a complex immune cell network that maintains the homeostasis required to fight pathogens that enter the microbiota. During the disease process, the lamina propria is invaded by innate immune cells, which include neutrophils, macrophages, natural killer cells, and adaptive immune cells like B-cells and T-cells. Innate immunity initiates when an antigen gets recognized by Toll-like receptors and nucleotide-binding and oligomerization domain (NOD)-like receptors located on the cytoplasm. Studies have shown the expression and function of both receptors are defective during the disease process. NOD2 mutations, commonly associated with Crohn’s disease, showed a weak ability to respond to lipopolysaccharide, which is said to contribute to disease severity. Evidence suggests that leads to decreased activation of nuclear factor kappa B (NF-κB), which leads to reduced anti-bacterial defense and increased microbial invasion and cause immune tolerance .Interleukins (IL)-23, a cytokine that influences innate and adaptive immunity, acts as an early defense mechanism against bacteria by inducing Th17 cells; polymorphisms of IL-23 revealed to be associated with the chronic inflammation seen in inflammatory bowel disease. The adaptive immune response consists of various types of T-helper cells  and regulatory T-cells, which are branches of CD4 T-cells secreting cytokines to maintain the homeostasis of the intestinal lumen. These branches of cells are critical against preventing entry to pathogens; however, reports show that the overexpression of these cells can contribute to the widespread inflammation seen in the disease process ,23.All findings come back to the importance of gut microbiota and inflammation for the pathogenesis of inflammatory bowel disease. Evidence suggests that altered gut microbiota can increase the permeability of the intestine, triggering inflammation as the etiology shared by both conditions. As mentioned, obesity and metabolic syndrome due to the diseased gut microbiota both contribute to the development of coronary artery disease.Atherosclerotic Plaques in Response to the Inflammatory ProcessAtherosclerosis is an inflammatory process responsible for many complications, including coronary artery disease, myocardial infarction, and peripheral arterial disease. Immune and adaptive cells regulate the process through proatherogenic inflammatory and anti-atherogenic mediators. Mononuclear phagocytes are present in all stages of the disease, illustrating the evidence to associate inflammation and atherosclerosis ,24.Studies thoroughly document no single marker can alone identify or diagnose inflammatory bowel disease. The serological markers best correlated with the inflammatory process include C-reactive protein (CRP), tumor necrosis factor-alpha (TNFα), immunoglobulins , vascular endothelial growth factor (VEGF), interleukin 1 (IL-1), and antineutrophil cytoplasmic antibody. The inflammatory cytokines after activation, regulate atherosclerotic plaque formation through increased oxidative stress, mononuclear phagocyte accumulation, and endothelial dysfunction .Several studies indicate C-reactive protein (CRP), an acute-phase reactant, and high sensitivity C-reactive protein (hs-CRP) are strongly associated with an increased risk of myocardial infarction. According to a meta-analysis done by Emerging Risk Factors Collaboration, CRP concentration showed a positive linear association with several conventional risk factors assessing for risk of vascular disease. Additional studies indicate increased CRP levels equated with relapse of the disease process. High CRP, with increased stool frequency and low serum albumin levels, was associated with a deterioration of ulcerative colitis and failure in medical therapy. At the same time, the American Heart Association and American College of Cardiology considered hs-CRP ≥2.0 mg/L as a strong risk factor for coronary heart disease, and recommend lifestyle modifications to reduce that the risk. In the intestinal immune system, macrophages have anti-inflammatory properties that remove bacteria with minimal damage. Disruption of this process leads to inflammation, impaired immune response, and increased growth of bacteria with filamentous and flagellated features. The flagellated bacteria begin an exaggerated inflammatory episode disrupting the intestinal mucosal barrier. As a result, lipopolysaccharide can cross the intestinal barrier triggering atherosclerosis , 25, 26.Inflammation, Thrombosis and Coronary DiseaseImmune-mediated disorders have been linked to increased cardiovascular events. Proposed mechanisms include autoantibodies, autoantigens, and inflammation triggering the synthesis and rupture of atherosclerotic plaque ,27-28.Studies show inflammation inhibits fibrinolysis, expresses procoagulants, and suppresses anticoagulants promoting thrombosis formation. Arterial and venous thromboembolism as a result of inflammatory bowel disease or flares has been well documented. Deep vein thrombosis and pulmonary embolism are the most prevalent complications arising from the hypercoagulable state of IBD, causing a high fatality. Endothelial injury, alterations in the clotting system, and venous stasis are the proposed mechanisms for thrombogenesis. Elevated plasma homocysteine levels have been seen in IBD patients. Homocysteine has been found to contribute atheroma and thrombosis formation once it binds to the endothelium -31. Inflammation being the driving force for IBD’s hypercoagulable state and atherosclerosis provides a lot of insight and further evidence that IBD can predispose to Myocardial Infarction.Common risk factors for IBD, MI, and coronary diseaseMany meta-analyses and cohort studies have been conducted globally to determine the association between IBD and MI. According to a study using population data from Finland, there was a notable increase in myocardial infarction in patients with IBD . A meta-analysis conducted by Feng and his team in 2017 demonstrated an increase in the risk of ischemic heart disease in patients with IBD, especially Crohn’s disease . Data alMost literature suggests trapped oxidized lipoproteins in the endothelium activate cells leading to the accumulation of chemokines and cell adhesion molecules, which signal monocytes to marginate and transform to foam cells by accumulating lipids which form the plaque. The same process was seen when microvascular endothelial cells were isolated from the intestine, suggesting shared environmental factors may predispose to the disease process of atherosclerosis leading to MI and IBD. A large retrospective study evaluating the efficacy of statins against inflammation illustrated an 18% decrease in the initiation of corticosteroid therapy. Routinely prescribed cardiovascular medications like statins showed decreased disease activity in IBD, suggesting further evidence on the link between IBD and MI ,33-36. Not all literature accepts the association between IBD and MI. According to a study using the nationwide inpatient sample, patients with IBD were less likely to be hospitalized with MI compared to the general population. The frequency of acute MI in IBD was compared to the rate it occurs in other immune-mediated disorders. A lower frequency was noted in IBD, indicating a difference in pathogenesis .Low-density lipoprotein (LDL) is a well-established marker for predicting CAD; in contrast, high-density lipoprotein (HDL) is known to exert anti-atherogenic and anti-inflammatory effects to prevent it. It is said that HDL can become proatherogenic due to inflammation, diabetes, and oxidative stress .According to a study done by Mitra et al. in 2015 indicated asymptomatic atherosclerotic plaques were associated with an increase of microorganisms of the gut microbiome, such as Bacteriodaceae. Streptococcacea, and Micrococcacea, while symptomatic plaques illustrated an abundance of Helicobacteracea and Neisseriaceae . Recent studies depicted that alteration of gut microbiota and its metabolites predisposed to hypertension and vascular pathology, as well as its ability to cause hypercholesteremia. Trimethylamine N-oxide (TMAO), a metabolite produced by the microbes, illustrated a positive correlation influencing the pathogenesis of MI. TMAO inhibits reverse cholesterol transport and accumulates foam cells (macrophages), which accelerate atherosclerosis ,40,41. AThis study does have some limitations. Only literature from the year 2000 was used to contribute to this review. A few articles contained only abstracts without the full text, depriving some specific examples of studies. Evidence in recent years is starting to endorse the association gut microbiota and pathogenesis of CAD. Although human trials were done, some of the studies showing the association between IBD and MI is based on experiments done on animals like mice. Most human research consisted of cross-sectional studies rather than longitudinal cohort studies. Mouse models usually analyzed one specific gene or function, but when it comes to humans, disruption of multiple genes and functions contribute to the disease process. Hence future researchers should focus on conducting additional cohort studies and clinical trials targeting the diseased gut microbiota of humans. After reviewing the literature, much evidence suggests inflammatory bowel disease (IBD) can predispose to myocardial infarction. Physicians should emphasize screening all young patients, particularly women under the age of 50, with IBD, as they have shown a higher risk of developing MI in IBD compared to men. Physicians should consider prescribing statins during the active disease process and counsel patients on lifestyle modifications to prevent the traditional risk factors for coronary artery disease. Future research should focus on clinical trials and population studies studying the diseased gut microbiota in patients. Evidence points to the altered gut microbiota, increasing intestinal permeability triggering inflammation as the pathogenesis shared by both conditions. Scientists should consider future research on performing more clinical trials in patients with anti-inflammatory medications to assess therapeutic efficacy in atherosclerosis. Drugs targeting diseased gut microbiota should be thoroughly studied as they could be the future in the management of atherosclerosis and inflammatory bowel disease.


Drosophila genome. We next performed a targeted RNAi screen aimed to identify the functional contribution of these different ion channels that may participate in excitation-secretion coupling in AKH producing cells (APCs). We assessed starvation survival, because changes in AKH signaling have previously been shown to impact starvation sensitivity. Genetic knockdown of three genes , in AKH producing cells caused highly significant changes (P < 0.001) in both male and female lifespan, and knockdown of six other genes  caused significant changes (P < 0.05) in only female lifespan. Specifically, the genetic knockdown of Ca-Beta and Sur led to increases in starvation lifespan, whereas the knockdown of sei decreased starvation survivorship. Focusing on these three strongest candidates from the behavioral screen, we assessed other AKH-dependent phenotypes. The AKH hormone is required for starvation-induced hyperactivity, and we found that these three ion channel gene knockdowns changed activity profiles and further suggest a modulatory role of these channels in AKH release. We eliminated the possibility that these genetic elements caused AKH cell lethality, and using independent methods, we verified expression of these genes in AKH cells. Collectively, these results suggest a model of AKH-cell excitability and establish an experimental framework for evaluating intrinsic mechanisms of AKH release.Adipokinetic Hormone (AKH) is the primary insect hormone that mobilizes stored energy and is functional equivalent to mammalian glucagon. While most studies have focused on exploring the functional roles of AKH, relatively little is known about how AKH secretion is regulated. We assessed the AKH cell transcriptome and mined the data set for specific insight into the identities of different ion channels expressed in this cell lineage. We found reliable expression of multiple ion channel genes with multiple members for each ionic species. Specifically, we found significant signals for 39 of the either known or suspected ion channel genes within the  Drosophila, which is paramount in regulating metabolism.In excitable tissues, changes in membrane potential are paramount in mediating specific aspects of cell physiology, including muscle contraction and hormonal secretion. A major molecular component that alters membrane potential are ion channels. Ion channels display considerable variation in their activation kinetics, their gating mechanisms, the specific ionic species that can pass . HoweverDrosophila, AKHR is also expressed in a small subset of central neurons, where is thought to facilitate gustatory sensitivity  is the functional equivalent of mammalian glucagon, as it is the primary hormone that facilitates the mobilization of stored energy . During sitivity , as wellsitivity .Drosophila, targeted ablation or blockade of hormonal release of AKH-expressing neuroendocrine cells cause increased survival during starvation , and the vast majority having a consistently low level of expression. Given the molecular stability of ion channel proteins, low levels of transcript expression are expected and consistent with other transcriptome analyses in Drosophila  and the Sulfonylurea receptor (Sur) gene caused a highly significant  increase in lifespan during starvation. Conversely, expression of an RNAi element targeting the seizure (sei) gene caused a highly significant reduction in starvation lifespan in both males and females as compared to control animals (AKH-GFP) (P < 0.001) (P < 0.05) deviated from our control line and include cac, stj, NaCP60E, Ih, TASK6, and Shaw .We evaluated starvation longevity in progeny of the crosses introducing an RNAi element to AKH cells. Introduction of RNAi elements targeting the < 0.001) . Notablyand Shaw . We alsoP < 0.001)  . Next, warvation .One potential explanation for the behavioral phenotypes is that these RNAi elements may adversely affect AKH cell viability or impair hormone synthesis. To evaluate potential AKH cell pathology associated with RNAi element expression, we employed a specific antibody against AKH to assess cell survival and hormone expression. We found that in all genotypes, AKH cell viability and minimally, synthesis of the AKH precursor were unaffected . These rHere, we present evidence that multiple ion channel genes are expressed in the neuroendocrine cells that release the Adipokinetic Hormone. Multiple ion channel transcripts were identified through an analysis of the AKH-cell specific transcriptome. We then employed a genetic approach to determine the overall contribution of these ion channels in modulating AKH secretion through the examination of AKH related starvation phenotypes. We identified that the genetic knockdown of three ion channel encoding genes strongly impacted starvation survival and found several other genes whose impacts were significant but modest in comparison. We also examined locomotor activity from animals expressing an RNAi targeting each of these three ion channel genes whose AKH cell specific knockdown highly impacted starvation survival. In each case, changes in locomotor activity were present either in the replete or starvation state. Lastly, we verified the knockdown of these genes did not cause AKH cell lethality, and we employed independent measures to confirm the AKH cell specific expression of these genes.Drosophila genome are expressed in this secretory tissue. However, we note that single cell (sc) transcriptomes of individual neurons also show a diversity of ion channels present  that are strong candidates in the regulation excitation-secretion coupling in this cell lineage (Ca-Beta) and despite the observation of all known pore-forming calcium channel genes being present in AKH cells via the transcriptome, knockdown of those produced largely no effect. We do note that the knockdown of cac, which is a pore forming calcium channel subunit , but only in females. We suspect that the behavioral output (starvation lifespan) has greater resolution in females as they live considerably longer during this challenge, as opposed to an inherent dimorphism mapping to AKH cells.Following our transcriptome analysis, we performed a targeted RNAi screen using AKH-dependent behavioral phenotype and identified three genes  were microdissected in HL-3 solution containing Triton X 100  and aspiDrosophila melanogaster genome BDGP6.22 using star v2.5 (p > 0.05). The source code for this analysis is available at github  in vials with a two percent agar solution to starve the animals . We asseLocomotor activity was monitored with a TriKinetics Locomotor Population Monitor  on the aggregate population of 30, 3–5 day old flies . Each viAKH-GAL4 transgene crossed to the UAS-RNAi lines were dissected. Brains were fixed in a 4% Paraformaldehyde/7% picric acid fixative for 1 h at room temperature and washed six times with phosphate buffered saline (PBS) containing Triton-X 100. A 1:1,000 dilution of polyclonal anti-AKH : ATACAATCAATCATCCGTCACAAC(reverse): TTGATCAAACGCTGTAAAACCTTASur : (forward) TTCAAAAGTTCTACAGGTGCTCAG(reverse): AAGGATTTCGGTGAATCTAGTCTGsei: (forward): AGGATCCCAATGACATGATTACTT(reverse): AATTGGATCGGAGTTGATGTATTThttps://www.ncbi.nlm.nih.gov/, PRJNA642982.The datasets generated for this study can be found in the online repositories. The names of the repository/repositories and accession number(s) can be found below: CS and EJ: design of experiments and analyzing data. CS, RP, JN, MR, and JB: performing experiments. EJ: drafting the manuscript. All authors contributed to the article and approved the submitted version.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.


Can prediction of suicide attempts after psychiatric hospitalization be improved using frequent assessments of the level and variability of an individual’s suicidal thoughts?In this prognostic study of 83 adult psychiatric inpatients, prediction of posthospital suicide attempts was fair when using only baseline data, improved in a model using mean level of suicidal thinking during hospitalization, and improved further in a model including dynamic features of suicidal thoughts.These findings suggest that data on real-time, dynamic changes in suicidal thoughts could improve prediction of suicide attempts during the high-risk period following psychiatric hospitalization. This prognostic study tests whether modeling dynamic changes in real-time suicidal thoughts during psychiatric hospitalization can improve predictions of postdischarge suicide attempts vs using only baseline data or using the mean level of real-time suicidal thoughts during hospitalization. The weeks following discharge from psychiatric hospitalization are the highest-risk period for suicide attempts. Real-time monitoring of suicidal thoughts via smartphone prompts may be more indicative of short-term risk than a single, cross-sectional assessment.To test whether modeling dynamic changes in real-time suicidal thoughts during psychiatric hospitalization can improve predictions of postdischarge suicide attempts vs using only baseline  data or using the mean level of real-time suicidal thoughts during hospitalization.In this prognostic study, 83 adults recruited from the inpatient psychiatric unit at Massachusetts General Hospital completed ecological momentary assessment surveys of suicidal thinking 4 to 6 times per day during hospitalization as well as brief follow-up surveys assessing suicide attempts at 2 and 4 weeks after discharge. Participants completed at least 3 real-time monitoring surveys. Inclusion criteria included hospitalization for suicidal thoughts and/or behaviors and English fluency. Data were collected from January 2016 to December 2018 and analyzed from January to December 2020.The primary outcome was suicide attempt in the month after discharge.Of 83 participants , 9 (10.8%) made a suicide attempt in the month after discharge. Mean cross-validated AUC for elastic net models revealed predictive accuracy was fair for the model using baseline data , good for the model using the mean level of real-time suicidal thoughts during hospitalization , and best for the model using dynamic changes in real-time suicidal thoughts during hospitalization ; this pattern of results held for other classification metrics  and when using different cross-validation procedures. Features assessing rapid fluctuations in suicidal thinking emerged as the strongest predictors of posthospital suicide attempts. A final set of models incorporating percentage missingness further improved both the mean  and dynamic feature  models.In this study, collecting real-time data about suicidal thinking during the course of hospitalization significantly improved short-term prediction of posthospitalization suicide attempts. Models including dynamic changes in suicidal thinking over time yielded the best prediction; features that captured rapid changes in suicidal thoughts were particularly strong predictors. Survey noncompletion also emerged as an important predictor of posthospitalization suicide attempts. This risk is particularly heightened among patients admitted with suicidal thoughts or behaviors, with the rate of suicide for these patients being nearly 200 times the global suicide rate.5 One factor limiting our ability to predict posthospitalization suicide attempts is that few studies have observed individuals during these short-term, high-risk time windows. A 2017 meta-analysis of the past 50 years of suicide research6 revealed only 1% of predictive analyses used time windows of less than 30 days, usually with just 1 assessment in that window. These studies fail to capture the short-term dynamic nature of suicidal thinking and cannot provide data on short-term risk of suicide attempts, particularly those following hospitalization.Suicide is a leading cause of death, claiming more than 800 000 lives each year globally and more than 45 000 in the United States.10 These methods have revealed that suicidal thoughts are characterized by large within-person fluctuations.9 Moreover, prior retrospective work suggests that suicide attempts are preceded by large, rapid changes in suicidal thinking.12 However, no studies have tested whether real-time measurement of changes in suicidal thinking can prospectively predict imminent suicide attempts, which could inform resource allocation for patients most at risk, in a setting where care is already accessible.13 Currently, clinical decision-making regarding hospital discharges is informed by patients’ self-report of suicidal thinking during specific clinical assessments . Brief standardized assessments of suicidal thinking administered continuously throughout hospitalization may aid clinical decision-making by providing novel information about a patient’s likelihood of suicide attempt following discharge.Recent advances in real-time monitoring technology provide opportunities to address these gaps via finer-grained ecological momentary assessment (EMA) of changes in suicide risk over shorter timeframes.14 we also built a second series of models as an exploratory aim to examine whether using missingness as a predictor would further improve accuracy of the mean and dynamic feature models.To advance the understanding of suicide in this high-risk period, we tested several different approaches to predicting posthospital suicide attempts using the following approaches: (1) baseline data at inpatient admission, (2) mean level of suicidal thinking during hospitalization, and (3) dynamic patterns of suicidal thinking during hospitalization. We hypothesized that computing and modeling dynamic fluctuations in suicidal thinking would yield improved predictive accuracy beyond both traditional static approaches of assessing suicidal thoughts (baseline model) and modeling only the mean of real-time suicidal thoughts during hospitalization (mean model). In addition, given evidence suggesting that missing data about suicidal thoughts may be an important predictor of suicide attempts,TRIPOD) reporting guideline.15In this prognostic study, we aimed to develop a model predicting posthospital suicide attempts using EMA data. All study procedures were approved by the Massachusetts General Hospital institutional review board, and all participants provided written informed consent. This study followed the Transparent Reporting of a Multivariable Prediction Model for Individual Prognosis or Diagnosis  completed at least 3 EMA surveys and were included in current analyses.Inclusion criteria for the study were admission to the psychiatric inpatient unit due to suicidal thoughts and/or risk (determined by patient self-report and clinician judgment) and English fluency. Exclusion criteria included any factors impairing participant ability to provide informed consent or participate in study procedures . Having a compatible smartphone was not an inclusion criterion; we provided a loaner phone to participants if needed. Recruitment for this study occurred in 2 waves, with 45 participants recruited in wave 1 (January 2016 through January 2017) and 59 participants in wave 2 (May 2017 through December 2018). Both waves used identical study protocols (with the exception of EMA survey software and frequency of assessments); we pooled data from both waves in the current study.Participants who met inclusion criteria and provided written consent to be in the study completed a baseline assessment during hospital admission. Participants then completed an EMA protocol for the duration of their hospital stay  with survey prompts sent to participants’ smartphones 4 to 6 times per day during waking hours. Participants were compensated $10 per day. Finally, participants completed follow-up assessments at 2 weeks and 4 weeks following discharge.16 We used the following characteristics for suicidal thoughts, plans, attempts, and nonsuicidal self-injury: lifetime presence, past-year frequency, past-month frequency, past-week frequency, and self-reported likelihood of future SITB engagement.Participants completed the self-report version of the Self-injurious Thoughts and Behaviors Interview (SITBI).17 to assess momentary suicidal thoughts on a scale from 0 (none) to 9 (very much). These were (1) desire to die by suicide (“How intense is your desire to kill yourself right now?”), (2) intent to die by suicide (“How strong is your intention to kill yourself right now?”), and (3) ability to resist the urge to die by suicide (“How strong is your ability to resist the urge to kill yourself right now?”).In wave 1, we used moviesensXS to send participants 4 semirandom prompts per day. In wave 2, we used Beiwe to send participants 6 semirandom prompts per day to obtain greater temporal granularity. Both waves used 3 items20 First, we used follow-up surveys at 2 and 4 weeks after discharge and categorized participants as having made a suicide attempt if they (1) reported hospital admission and responded “tried to kill yourself” to the question “What was the reason for your admission overnight to a hospital for mental health care?” or (2) responded yes to the question, “In the past 2 [4] weeks , did you make a suicide attempt ?” Second, we examined the electronic medical records of each participant to determine whether they returned to the hospital due to a suicide attempt in the month following discharge.We assessed presence of a suicide attempt in the month after hospitalization with 2 methods, which is common in longitudinal suicide research.21 via tsfeaturex,22 caret,23 and glmnet24 packages. We built 3 elastic net models  predicting posthospital suicide attempts, using the default threshold of 0.5 in glmnet. We used the elastic net algorithm, given substantial evidence that penalization improves prediction accuracy,27 alongside its well-established accuracy and robustness29 and ability to maintain clinical interpretability compared with less transparent machine learning algorithms .Analyses were performed in R version 3.6.1 30For the baseline model, we used the 20 baseline SITBI characteristics as predictors; 2.16% of data was missing and imputed with K–nearest neighbor imputation. The mean model included the mean of each EMA suicide item  as predictors. For the dynamic feature model, we extracted 24 time-series features for each EMA suicide item for each participant as predictors. These features characterize magnitude, speed, and probability of changes in suicidal thoughts over time, including the minimum, maximum, mean, standard deviation, percentage unique values , mean square successive difference , probability of acute change , and maximum change . Acute changes were defined as any successive change from an EMA prompt to the next in the 90th percentile or greater of the total distribution of change for each participant .31 we used 5-fold cross-validation with 3 repetitions to select the optimal λ (shrinkage) and α (mixing) parameters for each elastic net model. This also allowed us to obtain a cross-validation estimate of model performance. Whereas splitting a data set into a single training and testing set can have limited ability to accurately characterize uncertainty in results, particularly for smaller samples, repeating the training and testing process can provide more reasonable estimates of model accuracy in predicting outcomes in new data sets. Finally, we evaluated variable importance for all models using the varImp function in caret and extracted coefficients for the best model. Because no null hypothesis statistical testing was preformed, no prespecified level of statistical significance was set.Following recommendations by Kuhn and Johnson,Our primary model performance metric was the mean cross-validation estimate of area under the curve (AUC), a discrimination metric that ranges from 0.5 (chance) to 1.0 (perfect). We also evaluated several other metrics, including the mean cross-validated area under the precision-recall curve (AUPRC), accuracy, positive predictive value (PPV), sensitivity, specificity, Brier score, and Cohen κ. Higher scores for each of these metrics indicate better model performance, with the exception of Brier score, for which lower scores indicate better model performance.Primary models with EMA data were built using all available data with no imputation of missing EMA data. As a secondary aim, we fit elastic net models using missingness  as an additional predictor for the mean and dynamic feature model. To evaluate model robustness, we also fit our 3 primary models  using 2 different cross-validation procedures, ie, 10-fold repeated 3 times and leave-one-out-cross-validation.Participants’ demographic characteristics, diagnoses, and history of suicidal thoughts and behaviors on hospital admission are presented in the Model performance metrics for the baseline, mean, and dynamic models are presented in Variable importance metrics  for the dynamic model are shown in When using different cross-validation procedures to evaluate model robustness, results largely remained unchanged. For both 10-fold cross-validation and leave-one-out-cross-validation, the baseline model provided poor or fair performance, the mean model provided better performance, and the dynamic feature model performed the best  and dynamic feature  models . In thesThere were 3 key findings in this study. First, using EMA data greatly improved prediction of posthospital suicide attempts beyond traditional approaches, and models including dynamic changes over time performed particularly well. Second, the strongest predictors were probability of acute change in suicidal thoughts or intentions. Third, missingness greatly increased predictive accuracy for both the mean and dynamic feature models. Each of these findings warrants further comment.32 from 2019 suggesting that metrics of variability do not meaningfully predict mental health symptoms beyond central tendency . There are several potential explanations for this discrepancy, including that this earlier work focused on affect dynamics, whereas we examined fluctuations in suicidal thinking. Furthermore, dynamic metrics may be more sensitive to acute perturbations .33 In addition, most previous work has focused on relatively healthy individuals, whereas participants in the current study were hospitalized for a recent suicide attempt or severe suicidal thoughts. It is possible that, in acutely suicidal patients, variability in suicidal thinking provides more information about suicide risk than average levels of suicidal thinking. This is supported by research on the importance of affective instability in other psychiatric populations . Future research examining the benefits of temporally granular measurement and modeling dynamic features to predict suicide attempts could shed additional light on these important questions.Model performance for the mean and dynamic feature models was substantially better than that for the static baseline model across all classification metrics. This suggests that collecting EMA data to obtain more temporal granularity in the measurement of suicidal thoughts can enhance our ability to predict suicide attempts. The dynamic feature model provided higher average AUC than the mean model, suggesting metrics of variability may be more important than central tendency. Indeed, variable importance metrics from the dynamic feature model indicated that the 3 most important predictors were probability of acute change in the desire to kill oneself, intent to kill oneself, and ability to resist the urge to kill oneself. Given that probability of acute change indexes the likelihood of an individual experiencing extreme shifts in suicidal thinking, rapid fluctuations in suicidal thinking may increase the risk of suicidal behaviors beyond average levels of suicidal thinking. This finding contrasts meta-analytic work36 Although machine learning methods have yielded high AUCs, the PPV of most models has been low. A 2019 review of 64 longitudinal suicide prediction models36 found most models reported extremely low  PPVs, such that 99 out of every 100 individuals predicted by these models to attempt suicide will not. In comparison, the average cross-validated PPV for our dynamic feature model was 0.39. This represents a substantial improvement beyond both previous work and our baseline and mean models  and was likely influenced by the relatively high base rate of suicide attempts in our sample. Prediction models with low PPVs can still have a positive net benefit if the benefits of interventions with true-positive results outweigh costs of intervening with false-positive results.37 Future studies with larger sample sizes are needed to replicate these results and determine whether models using EMA data can provide acceptable AUC and PPV scores to guide clinical decision-making and suicide prevention efforts.The dynamic feature model predicted posthospital suicide attempts with good accuracy , which is at the high end of the range of reported AUC values for longitudinal prediction models of suicidal behaviors.14 and highlight the importance of future studies taking an informative missingness approach for predicting suicidal behavior. However, caution is warranted in interpreting these findings, given that some evidence suggests using missingness for predictive modeling may introduce bias into models and generalize poorly to new data sets.38As a secondary aim to explore the importance of missing EMA data, we added EMA missingness  as a predictor to both our mean and dynamic feature models, which greatly enhanced performance of both models . Notably, this missingness variable also emerged as the top predictor in the mean model and the second most important predictor in the dynamic feature model. These findings are consistent with prior work finding that nonresponse to questions about suicidal thinking is a particularly strong predictor of the transition from suicidal thinking to suicide attempts39 could shed further light on the potential for effective risk detection in hospitals using EMA. Alongside future work on effective interventions and clinical guidelines, this could provide important information to identify and provide patients with support in a setting where care is already available and accessible.These findings should be considered in the context of several limitations. First, we had a relatively small sample size (83 participants) that was predominantly White (83.1%). Future studies would benefit from larger sample sizes and longer follow-up periods, particularly given that suicide attempts are a low base-rate behavior, as well as more diverse and representative samples. Second, we only included self-reported measures of suicidal thoughts and urges; future research incorporating other self-reported measures  alongside psychophysiological and smartphone data  may produce stronger models. Third, although the within-sample design of this study allowed for more than 1300 individual observations, we had a relatively small number of observations for some participants . This was partly due to the intentional naturalistic design of the study, with participants completing EMA prompts only for the duration of their hospital stay, which differed in length for participants. Nonetheless, overall compliance rate was relatively low , and many missing responses may not have occurred at random. For instance, it is possible that participants did not complete responses when they were meeting with members of their care team  or were highly distressed. Finally, only 65 of the original 83 participants completed a follow-up survey, and we did not have access to claims and/or admissions data for all other hospitals in the area. Replicating these findings and models with more stringent compliance protocolsThe current study provides novel insight on the high-risk period immediately following discharge from psychiatric hospitalization. These findings suggest that collecting dynamic, real-time data from patients during hospitalization can significantly improve the prediction of posthospitalization suicide attempts. Such information could be vital in clinical decision-making about discharge and posthospital level of care. Continued research incorporating temporally granular assessments of suicidal thoughts during high-risk time periods may advance the understanding, prediction, and prevention of suicide.


Clinical results with oncolytic adenoviruses (OAds) used as antitumor monotherapies show limited efficacy. To increase OAd potency, transgenes have been inserted into their genome, a strategy known as “arming OAds”. Here, we review different parameters that affect the outcome of armed OAds. Recombinant adenovirus used in gene therapy and vaccination have been the basis for the design of armed OAds. Hence, early region 1 (E1) and early region 3 (E3) have been the most commonly used transgene insertion sites, along with partially or complete E3 deletions. Besides transgene location and orientation, transcriptional control elements, transgene function, either virocentric or immunocentric, and even the codons encoding it, greatly impact on transgene levels and virus fitness. In the 1950s–1970s, anecdotal reports of spontaneous tumor remissions as a result of naturally acquired viral infections prompted the clinical concept of using pathogenic viruses to cure cancer, known as virotherapy. One of the first clinical trials was done in 1956 using wild-type adenovirus (Ads) as a therapeutic agent in cervical cancer. It was demonstrated to be safe with mild flu-like symptoms as the main adverse effects [In 1996 Frank MacCormick proposed the use of a mutant adenovirus to treat p53-deficient tumors . In the To increase the efficacy of oncolytic adenoviruses major research efforts have been directed to enhance tumor-targeting by capsid modifications, and tumor-selective replication using promoters and gene deletions. These aspects have been broadly reviewed in the literature ,5,6. RepAdenovirus has several advantages compared to other oncolytic viruses for transgene insertion. All RNA viruses except orthomyxoviruses  and retroviruses replicate in the cytoplasm and therefore nuclear transcriptional control elements, such as tumor selective promoters, are non-functional. In contrast, DNA viruses except poxviruses  replicate in the nucleus and are compatible with the use of eukaryotic promoters. Viruses with small compact genomes cannot accept transgenes . Besides, adenovirus stands out from the rest of transgene-carrying viruses  because it has a highly regulated temporal sequence of viral gene expression starting with E1a which allows tuning transgene expression.Here we review the different strategies used to arm oncolytic adenoviruses. Although the nature of the therapeutic transgene would be the first important feature for an armed-OAd, it is crucial to note that the amount, and timing of transgene expression are pivotal for this arming strategy. In this review, we explore how transgene location, different transcriptional control elements, the transgene codon usage, and the transgene itself affect its expression, kinetics, and viral replication.Adenoviridae is a family of icosahedral, non-enveloped viruses with an approximately 30–40 kb linear double-stranded DNA genome. In humans, more than 50 different serotypes associated in six groups (A to F) have been discovered. Nevertheless, the most common for oncolytic therapy are serotypes Ad2 and Ad5 from group C. These are the best characterized among all the serotypes; replicate to higher yields than most other human Ad serotypes, and DNA sequences and tools are readily available for their genetic modification .Adenoviruses maximize their genome coding capacity by generating early and late transcription units  controlled by multiple promoters, which in turn generate different RNAs and proteins by alternative splicing ,15 Figu. The strcytosine deaminase gene (CD) fused to the herpes virus thymidine kinase (TK) (thymidine kinase (TK) gene was inserted in an E1b55K and E3-deleted adenovirus after the E1 region [In contrast to gene therapy vectors, OAds replicate in cancer cells. Thus E1 cannot be deleted entirely. The partial deletion of E1b55K was the first deletion postulated to be dispensable in tumor cells, based on the rationale that the p53-blocking function of this protein is not needed in p53-deficient cells . Freytagase (TK) . This vi1 region . Since tE3 gene [To bypass the need of exogenous promoters in OAds the group led by Terry Hermiston at Onyx substituted E3 genes with transgenes maintaining the endogenous promoters and polyadenylation sites, and endowing the transgene with the expression kinetics of the substituted E3 gene ,27,28. TE3 gene . HoweverE3 gene ,30. The E3 gene . Hence, E3 gene ,33,34.The Hermiston group also published the replacement of E3B adenoviral genes , splice acceptor sites (SA) or protein fusions using 2A self-cleaving peptide linkers . This stAlternatively, microRNAs (miRNA) can be used to regulate transgene expression. They are short-length non-coding RNAs that bind to complementary target sequences, leading to suppression of gene expression via post-transcriptional regulation. Their use to restrict OAds replication has been reviewed . MoreoveExogenous promoters have been extensively used in adenovirus vectors for gene therapy and they were used in the first armed OAds. The most widely used promoter is the human cytomegalovirus promoter (CMVp), a constitutive promoter trans-activated by early adenoviral proteins but easily silenced in mammalian systems. CMVp confers high levels of transgene transcription at early timepoints independent of virus replication. Transgenes are transcribed in every infected cell, even in normal tissues. To achieve tumor-selective expression, transgene transcription has been controlled by cellular promoters overactive in tumor cells. For instance, an OAd encoding GFP controlled by a fusion promoter between human telomerase reverse transcriptase (hTERT) and a small fragment of the CMVp . ProgresThe main limitation of exogenous promoters is that they require large DNA fragments, increasing the size of the transgene cassette, and thus compromising the packaging limit of OAds. Moreover, the transgene kinetics could not be carefully controlled and becomes independent of virus replication.Internal ribosome entry sites (IRESs) are highly structured viral sequences that facilitate biscistronic gene expression by internal RNA translation initiation in addition to the cap-dependent translation at the 5′ of mRNAs. In consequence, IRES allow trangene expression with the same kinetics as the linked viral gene. When connected to a late gene, transgene expression can be restricted to the sites of virus replication, which is tumor-selective in the case of OAds. The IRES sequence from the encephalomyocarditis virus was used to express transgenes from early and late Ad transcription units, obtaining higher transgene levels than CMV-armed OAds at late stages ,58,59,60The viral 2A sequences are short peptide sequences that facilitate multiprotein expression from one open reading frame by a process termed ribosomal skipping ,62. Its Alternative splicing was first described for adenovirus late mRNAs  and it ccytosine deaminase gene after Ad5 adenoviral fiber [beta globulin gene (branch point and splice acceptor sequence (BPSA) has been used in the adenoviral context to express transgenes [The IIIa splicing unit and splice enhancer (3VDE) was the first one to be studied in detail . We haveal fiber . Alternaal fiber . An artiansgenes . It was ansgenes . Howeveransgenes  and Luciansgenes , the IREansgenes .Several transgene features have to be considered when arming OAds. As mentioned above, the length of the transgene has a major impact on the design of armed OAds. The insertion of transgenes longer than 2kb requires deletions in the adenoviral backbone to be adequately packaged into virions. In these cases, saving genome space to avoid excessive adenoviral gene deletions is crucial, as we have seen with the detrimental in vivo impact of E3 deletions. Thus, embedding the transgene into deleted transcription units taking advantage of endogenous promoters or the use of short sequences such as SA is the best option to control transgene transcription.The transgene sequence may impact on the arming outcome at different levels. Eukaryotic transgenes may present introns in their sequences that are important for post-transcriptional stability and nucleocytoplasmic transport. Indeed, an increase of transgene expression in vitro and in vivo was reported in gene therapy vectors using introns in the expression cassette . Howeverfiber gene sequence to the human codon usage in an adenovirus decreased fiber expression and reduced expression of other structural proteins such as hexon and penton, compromising viral fitness. Furthermore, the introduction of GFP as a late gene after fiber under a splicing acceptor further reduced viral fitness [The set of codons encoding the transgene, known as the codon usage, might also affect its expression and viral fitness. We reported that adenovirus proteins could be grouped according to their codon usage. In particular, most early viral regulatory proteins used codons with A/T ending, while proteins implicated in replication and virion formation, including structural proteins, used codons ended in G/C. However, the fiber presented a different codon usage compared to the rest of the structural proteins, which was also poorly adapted to the human codon usage. Optimization of the Ad5  fitness .According to these results, changing a given protein codon usage, such as the fiber, could generate an imbalance in aminoacylated-tRNA (aa-tRNA) use between structural proteins resulting in a competition for the same aa-tRNA host pool leading to decreased late protein expression and viral fitness. This could explain why the insertion of a transgene as a late gene, in this case GFP, with a codon usage competing with structural proteins further attenuated virus replication. The results suggest that transgene codon usage should be adapted to avoid competing with the aa-tRNA frequently used by late genes, a feature to consider when arming OAds.The level of transgene needed for the therapeutic effect or the potential cytotoxicity of the transgene should be taken into account for the selection of the appropriate transgene location or transcriptional control elements. For instance, non-cytotoxic transgenes could be encoded in early regions with constitutive promoters or linked to adenoviral late genes. Otherwise, cytotoxic transgenes that may hamper virus-producer cells before completing the viral replication cycle should be encoded in late phases and at lower amounts to allow proper virus replication. The constitutive or immediately early phase expression does not restrict the transgene transcription to the tumor and all infected cells produce transgene, independently of virus replication. Regarding this, toxicity in vivo has to be addressed, especially in systemic administrations.Therefore, transgene function is a key feature for arming design. The function of transgenes can be considered virocentric or immunocentric. Virocentric transgenes would enhance virus cytotoxicity, yields or spread. Immunocentric transgenes aim to enhance the immune responses elicited during the oncolysis. The rationale behind immunocentric arming is that anti-tumor immune response may be the ultimate mechanism of efficacy for OAds. Here, we review some examples of both types of transgenes and summarize the armed-OAds in clinics.Transgenes aiming to improve the oncolytic effect, enhance virus spread, or kill bystander non-infected cells can be considered virocentric. Initially, genes used to arm OAds were prodrug-converting enzymes for molecular chemotherapy, known as suicide genes. These enzymes promote the conversion of intravenously administered non-toxic prodrugs to toxic drugs in adenovirus-infected cells, resulting in high local concentrations in the tumor. They induce a bystander effect of the toxic drug with limited systemic toxicity. Some examples of commonly used enzymes are herpes simplex virus thymidine kinase (TK) , bacteriFollowing this rationale, toxins were also used to kill surrounding non-infected cells. For instance, an OAd was armed with melittin, a water-soluble toxic peptide from the bee venom that induces cell apoptosis ,83. ToxiAnother virocentric approach is to enhance the viral release and spread of OAds. Apoptosis stimulates adenoviral spread through apoptotic bodies . When apAnother strategy to increase the cytotoxicity of OAds is based on RNA interference (RNAi), recently reviewed . BrieflySPAM1) to degrade hyaluronic acid from the tumor matrix [Connective tissue, fibrosis, and extracellular matrix hamper viral spread in the tumor . OAds har matrix ,110,111.r matrix . Matrix Fusogenic membrane glycoproteins induce cell-cell fusion and massive syncytia formation. OAds have been armed with measles virus fusogenic membrane glycoprXoteins  or gibboIn the last decade, mounting evidence that the immune system can be a powerful ally to cure cancer, mostly fostered by the development of checkpoint inhibitors in oncology, has shifted the strategy in favor of immunocentrism. Oncolytic viruses are an excellent adjuvant for immunotherapy by debulking the tumor, releasing tumor antigens, and providing the appropriate danger signals to inflame tumors and make them susceptible to immunotherapy treatments. As a clear example of this trend, arming OAds with immune-stimulatory cytokines or redirecting immune molecules has emerged as the main approach to prevent cancer spread and recurrence by overcoming the immune tolerance in the tumor. OAds with these transgenes usually contain E3 deletions to provide space and eliminate E3 immune evading functions, and the transgene is expressed early or constitutively. This approach has been widely used, and it is suitable for the most of cases. However, if used systemically such a constitutive expression could lead to toxicity, and it might be appropriate to consider late phase tumor-selective expression.Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates the development, recruitment, activation, and survival of dendritic cells, which are vital for antitumor immunity. ONCOS-102  is an onInterleukin 12 (IL-12) is a heterodimeric cytokine that strongly promotes T-cell helper type 1 (Th1) responses by activating macrophages and dendritic cells. OAds with IL-12 alone or combined with other immune molecules showed antitumor activity even in murine models where human adenoviruses are not able to replicate ,123,124.The combination of immunostimulatory cytokines and immune ligands represent the latest trend. Arming with TNFα and IL-2 caused a Th1 enhanced immune response in preclinical models  and the Despite the unprecedented efficacy of immune checkpoint inhibitors (ICIs) for certain tumor types, lack of responses in many poorly inflamed (cold) tumors and the systemic autoimmune toxicity have prompted the use of OAds to deliver ICIs. An OAd producing a full-length human monoclonal antibody specific for CTLA4 was published for first time in 2012 . Later, Armed-OAds evolve in parallel to the immunotherapy landscape. OAds have been designed to produce soluble ligands of immune checkpoints such as OX40L , GITRL  or a fusThe best arming strategy depends on the transgene and the desired amount and timing of expression. We have reviewed all published genome insertions sites in oncolytic adenoviruses. Most armed OAds have E3 deletions, which reduces the virus persistence in vivo due to a lower immune-evasion. Thus, we postulated that E3 insertion site could be beneficial for immunocentric transgenes aiming to stimulate the immune system. On the other hand, we suggest that virocentric transgenes or those that need higher expression could be inserted at late sites in non-E3 deleted viruses for longer persistence in vivo, always considering the packaging limits of the virus. In terms of transcriptional control, taking advantage of the endogenous E3 promoters using gene replacements or the major late promoter using splice acceptors allows efficient expression with minimal genomic size increase. In particular the use of the major late promoter allows high expression linked to the virus replication.