Immune checkpoint inhibitors (ICIs) have shown definite therapeutic effects in various types of cancers, especially non-small cell lung cancer (NSCLC). However, ICIs have unique side effects, called immune-related adverse events (irAEs), which can occur in various systems throughout the body. Among such irAEs, immune checkpoint inhibitor-related pneumonitis (ICI-P) is a fatal adverse reaction. In this review, we discussed the risk factors, pathogenesis, clinical characteristics, radiological manifestations, pathological features, diagnosis, grading, and management of ICI-P in NSCLC and the relationship between ICI-P and the efficacy of ICI therapy. In addition, we discussed the predictive factors for ICI-P. This review will play a crucial role in the prediction, evaluation, and management of ICI-P for widespread application of immunotherapy. Lung cancer is one of the most common malignant tumours worldwide, with non-small cell lung cancer (NSCLC) accounting for 85% of cases , 2. AccoImmune checkpoint inhibitors (ICIs) exert significant clinical therapeutic effects and have accelerated the treatment of advanced cancer in the new era of immunotherapy. The use of ICIs has shown great success in improving the overall survival (OS) and progression-free survival (PFS) rates of NSCLC –7. ICIs IrAEs can affect all organs of the body, including the skin, gastrointestinal tract, liver, kidneys, lungs, endocrine organs, and the central nervous system –11. AmonMany potential factors can increase the incidence of ICI-P in NSCLC patients treated with ICIs. Among the 97 patients who participated in the subgroup analysis of the Keynote-001 trial, OS was significantly longer after pembrolizumab administration in patients who had previously received radiotherapy than in patients who had not received radiotherapy. However, 3 3/24, 13%) patients who underwent thoracic radiotherapy had developed ICI-P, whereas only 1  patient who did not receive thoracic radiotherapy had developed ICI-P. Therefore, the probability of treatment-related pulmonary toxicity is higher in patients who have received radiotherapy than in patients who have not received radiotherapy . Other c, 13% patAccording to a recent meta-analysis of ICI clinical trials, the total incidence of ICI-P after single and combination therapy is 1.6% and 6.6%, respectively, suggesting that the risk of ICI-P is higher after combination therapy than after single therapy . In anotA retrospective study enrolling 1826 patients with cancer reported that ICI-P occurred more frequently in men and former or current smokers (64 [3.5%] patients) . NakahamIn conclusion, patients with the following characteristics: male, former or current smoker, ≥65 years old, previous chest radiotherapy, previous lung disease, combination therapy, and TICA are predisposed to ICI-P after immunotherapy Table 2.The mechanisms of ICI-P remain unclear; however, some theories based on the mechanism of action of ICIs and related studies are described below. Activated T cells, B cells, NK T cells and myeloid cells express PD1 . Tumour ICI-P is a unique toxic reaction that occurs after immunotherapy. Although it is relatively rare, it is one of the most important causes of death caused by ICIs in patients with NSCLC. A meta-analysis by Mizuki Nishino  showed tICI-P is non-infectious pneumonitis, and its clinical manifestations are different from ordinary pneumonitis . A studyWhen ICI-P is suspected, clinicians should make accurate and rapid decisions, because ICI-P has characteristics that demand urgency in treatment. However, the clinical manifestations of ICI-P are diverse, and it is difficult to predict the occurrence of ICI-P before initiating treatment. CT of the chest plays a significant role in the diagnosis of ICI-P. Understanding the features of CT of the chest in ICI-P is important for prompt treatment. At present, the imaging classifications are mainly divided into the following categories: organising pneumonitis (OP), non-specific interstitial pneumonitis (NSIP), hypersensitivity pneumonitis (HP) and diffuse alveolar damage (DAD). DAD is also called acute respiratory distress syndrome (ARDS). The severity of these conditions is graded as follows: DAD>NSIP/HP>OP. In terms of incidence rate, OP has the highest incidence (65%), NSIP has a lower incidence (15%) and HP and DAD have the lowest incidence (10%) , 56. ThePathological methods are becoming increasingly essential for the diagnosis of ICI-P. They can be used to rule out infectious pneumonitis and tumour progression. However, BAL and lung biopsy are not routinely performed in patients with ICI-P. In a retrospective study by Brandon T on 9 patients with ICI-P , OP was The diagnosis of ICI-P requires a comprehensive consideration of the clinical symptoms, as well as general bloodwork, CT imaging, and invasive evaluation (BAL or lung biopsy). Exclusion diagnosis is also an important strategy. Infectious pneumonitis, radiation pneumonitis (RP), tumour progression, carcinomatous lymphangitis, and pulmonary oedema caused by heart failure or myocarditis are common differential diagnoses. Establishing a diagnosis of ICI-P requires the exclusion of diseases mentioned in According to the latest National Comprehensive Cancer Network (NCCN) guidelines, ICI-P is divided into three levels and four grades as follows: mild (G1), moderate (G2) and severe (G3-4) Table 4.Steroid therapy is a routine strategy for the management of ICI-P. Regular and sufficient use of steroids can help to treat 70—80% of patients with ICI-P . AccordiSome scholars have compared a large amount of research data and concluded that the occurrence of irAEs is directly proportional to the prognosis . The occThe incidence of serious ICI-P after immunotherapy adversely affects the survival and quality of life of patients. The discovery and optimisation of predictive factors through laboratory tests can help clinicians to predict the occurrence of ICI-P, discontinue the use of ICIs in time or administer steroids early and may help in prolonging the survival of patients. A study showed that the expression of PD-L2 may be related to the incidence of irAEs in patients with NSCLC treated with PD-1 inhibitors. In addition, pre-existing autoimmunity markers such as the rheumatoid factor have been identified as independent predictors of skin reactions caused by ICIs . ThyroidClinicians should be cognizant of adverse reactions caused by ICIs, especially ICI-P. It is important to make an assessment before administering medications and pay attention to high-risk groups. After the administration of immunotherapeutic drugs, clinicians should pay close attention to changes in the condition of patients. Based on the combination of clinical manifestations, imaging data, and pathological characteristics of patients, ICI-P can be easily diagnosed, considering that infectious pneumonitis is excluded. ICI-P is mild in most cases and can be cured by appropriate treatment such as discontinuing immunotherapy or using steroids. If severe ICI-P occurs, immunotherapy should be promptly discontinued, and steroids and immunosuppressive therapy should be administered. Early intervention has a great impact on the survival and quality of life of patients. More studies are required for steroid-refractory pneumonitis, because, at present, an effective standard treatment plan is not available. In addition, further investigation is warranted to identify the predictors of ICI-P in the future.All authors planned and wrote the manuscript and contributed to the article and approved of 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Glycans expand the structural complexity of proteins by several orders of magnitude, resulting in a tremendous analytical challenge when including them in biomedical research. Recent glycobiological research is painting a picture in which glycans represent a crucial structural and functional component of the majority of proteins, with alternative glycosylation of proteins and lipids being an important regulatory mechanism in many biological and pathological processes. Since interindividual differences in glycosylation are extensive, large studies are needed to map the structures and to understand the role of glycosylation in human (patho)physiology. Driven by these challenges, methods have emerged, which can tackle the complexity of glycosylation in thousands of samples, also known as high-throughput (HT) glycomics. For facile dissemination and implementation of HT glycomics technology, the sample preparation, analysis, as well as data mining, need to be stable over a long period of time (months/years), amenable to automation, and available to non-specialized laboratories. Current HT glycomics methods mainly focus on protein N-glycosylation and allow to extensively characterize this subset of the human glycome in large numbers of various biological samples. The ultimate goal in HT glycomics is to gain better knowledge and understanding of the complete human glycome using methods that are easy to adapt and implement in (basic) biomedical research. Aiming to promote wider use and development of HT glycomics, here, we present currently available, emerging, and prospective methods and some of their applications, revealing a largely unexplored molecular layer of the complexity of life. The surfaces of all cells are covered with a dense layer of glycans and nearly all proteins which evolved after the appearance of multicellular life are glycoproteins. This indicates the functional importance of glycans in many biological processes . AdditioN-glycans. In this perspectives paper, we describe glycan analysis methods that have been used in actual HT glycomics studies, that is, comprising thousands of samples and implementing automatable sample preparation and data analysis, and we discuss their advantages, limitations, and perspectives. As current HT glycomics methods mainly focus on protein N-glycosylation, this type of glycosylation is central for the current work. Additionally, the promises and challenges for the HT analysis of other types of glycoconjugates are highlighted.The polypeptide part of a protein is largely defined by a genetic template. The analysis of gene sequence variation and transcript levels are a good proxy to study variations in the proteome, but this is not the case with glycans. While glycosylation is considerably heritable , it is iWhile many classes of glycoconjugates exist, including glycoproteins, free oligosaccharides, proteoglycans, glycosaminoglycans, and glycosphingolipids , only glN-glycans—oligosaccharides attached to the Asn in an Asn-Xxx-Ser/Thr (Xxx ≠ Pro) motif—has matured significantly over the last decade, relying mostly on enzymes that allow the straightforward, largely unbiased, and non-destructive release of N-glycans from proteins. These enzymes are available for different classes of N-glycans, with PNGase F being most broadly used for human N-glycosylation and with e.g. PNGase A enabling cleavage of plant and insect N-glycans. Unfortunately, at the moment, no such tools are available for the other types of protein glycosylation.There are roughly three levels on which protein glycosylation can be assessed . The firO-GalNAc-type  glycopeptides are also reported, such as those covering the O-glycosylated hinge region of human immunoglobulin A1 (IgA1)  . GlycopeN-glycans as well as glycopeptides of isolated proteins have truly matured, these approaches, together with their indispensable data processing solutions, will be detailed in the following sections.Finally, intact glycoprotein analysis is emerging in the field of HT glycomics, which is so far only sparsely applied on larger sample sets. A notable example is the analysis of intact apolipoprotein CIII that harbors a single O-glycosylation site . In prinN-glycans include hydrophilic-interaction liquid chromatography (HILIC) with fluorescence detection (FLD), capillary gel electrophoresis (CGE) with laser-induced fluorescence (LIF), and matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS)   . From Fie 3-arm) . AlthougN-glycans  allows glycans to be separated based on their size, structure (monosaccharide composition and regioisomerism), and charge. Prior to HILIC–FLD analysis, released glycans are subjected to unbiased and uniform reducing-end labeling to introduce a fluorophore allowing FLD detection . Reducin-glycans . Accordi-glycans . Glycan CGE–LIF offers an alternative separation strategy for the analysis of released glycans based on differences in their charge-to-size ratio. Similar to HILIC–FLD, this approach allows the efficient separation of neutral and charged glycans in a single run and the partial separation of structural glycan isomers . The fluN-glycans, which can readily be performed on hundreds of samples with high precision using MALDI–MS  detection can support structural assignment. By contrast, online coupling of CGE–LIF is analytically challenging and not routinely achieved , an important player in adaptive immunity . WorkfloRecent technological developments yielded the sensitivity to analyze low abundance glycoproteins and to handle minute amounts of biological material. Stable low-flow ESI conditions of 100–10 nL/min greatly enhanced the ionization efficiency of glycopeptides . FurtherFollowing the strategy outlined above, LC–MS lends itself to challenging HT investigations, such as the study of antigen-specific antibodies  and antim/z values. For (LC–)MS data, tools are available, including Glycoworkbench automatic curation of individual analytes and measurements. In all separation-based approaches, including LC–MS, it is important to cover the complete chromatographic/electrophoretic peaks during quantification, which, in the case of variations in retention or migration times, requires alignment tools and accurate peak detection algorithms. This additional level of variation, next to the m/z dimension, makes the processing of glycoproteomics LC–MS data particularly challenging, and improved tools are needed to support curation of these complex datasets.Having established a list of target analytes, relative glycoform quantification can be achieved by HappyTools  for LC–FCE  for CGE–CE  for direCE , and SkyCE  for LC–MMost commonly, total area normalization—per protein and site, if available—is used to obtain the final relative quantification of glycoforms. The absolute quantification of released glycans and glycopeptides is still in its infancy and is not implemented in HT approaches. Absolute quantification in LC–FLD and CE–LIF would need consistent, close-to-complete glycan release and labeling. Current methods are not validated for these aspects. Regarding quantitative detection by MS, while isotopic labeled standards and labels are available and have shown applicability , challenGlycomics data are multivariate and it remains a challenge to give a comprehensive and intelligible overview of complex glycomes. For this purpose, automated visualization tools, such as Glynsight , have beN-glycome  randomization . ReplicaTo control for the effect of experimental factors on glycan measurements, the blocked randomization of samples between batches is critical. In such a design, every batch (block) maintains a constant distribution of known experimental and main biological factors . To alloN-glycans derived from a wide variety of liquid biopsies and isolated proteins. In addition, HT glycomics successfully targets N- and O-GalNAc-glycopeptides of a select set of glycoproteins . This strength is, for example, exploited in multiple attribute monitoring in biopharma . Fully aMS of intact proteins allows the analysis of glycosylation in a protein-specific way along with other protein structural features and PTMs. A big advantage of intact protein analysis is the often very simple sample preparation workflow, favoring the use of this approach in biopharma for batch release as well as in clinical diagnostics . For intN-glycan spatial distribution in tissues is currently investigated using advanced MS imaging (MSI) workflows. The spatial resolution of these methods is in the range of 5–20 μm and not yet advanced enough to study single cells  rocesses . Hence, rocesses . These dAlso, the type of samples targeted by HT glycomics should be extended in the future. While the current focus is mainly on liquid biopsies, with plasma and serum being most thoroughly explored, glycans are of great importance at the interface between cells or tissues. The optimization of analytical sensitivity, and sample preparation workflows targeting cell surface and tissue extracellular matrix glycomes, will allow the exploration of this mostly uncovered layer of the human glycome with, as holy grail, single-cell glycomics. Important factors in enhancing sensitivity are simplified and miniaturized sample preparation protocols in combination with ultra-low flow separation modules coupled to MS.N-glycans can indeed be altered with weight loss , the Centre of Competence in Molecular Diagnostics grant (#KK.01.2.2.03.0006), the Croatian National Centre of Research Excellence in Personalized Healthcare grant (#KK.01.1.1.01.0010), the Dutch Research Council project “Proteoform-resolved pharmacokinetics of biopharmaceuticals” (ENPPS.LIFT.019.012), and the European Research Council (ERC) project “GlycoSkin” .Conflict of interest statement: Maja Pučić-Baković, Mislav Novokmet, Genadij Razdorov, Frano Vučković, Irena Trbojević-Akmačić, and Maja Hanić are employed at, and Gordan Lauc is the founder of, Genos Ltd, a private research organization that specializes in HT glycomic analysis and has several patents in this field. Manfred Wuhrer is the inventor on a patent on MS glycan profiling: EP 3457123 A4.2-AB: 2-aminobenzamideAPTS: 1-aminopyrene-3,6,8-trisulfonic acidBMI: body mass indexCGE: capillary gel electrophoresisESI: electrospray ionizationFLD: fluorescence detectionFT-ICR: Fourier-transform ion cyclotron resonanceHILIC: hydrophilic-interaction liquid chromatographyHPLC: high-performance liquid chromatographyHT: high-throughputIg: immunoglobulinIgA: immunoglobulin AIgG: immunoglobulin GLIF: laser-induced fluorescenceMALDI: matrix-assisted laser desorption/ionizationMS: mass spectrometryMSI: mass spectrometry imagingPGC: porous graphitized carbonTOF: time-of-flightUHPLC: ultrahigh-performance liquid chromatography20220412_SupTables_cwac026Click here for additional data file.


Furthermore, this work shows in detail the specific adverse events during CAR-T cell therapy and the role of [18F]FDG PET/CT imaging in their occurrence.Chimeric antigen receptor–engineered T cells are an innovative therapy in hematologic malignancies, especially in patients with refractory/relapsed B-cell lymphomas. Few studies have analyzed the role of [18F]FDG PET/CT plays a key role during staging and response assessment in patients with lymphoma; however, the evidence about its utility in CAR-T therapies for lymphomas is limited. This review article aims to provide an overview of the role of PET/CT during CAR-T cell therapy in B-cell lymphomas, focusing on the prognostic value of metabolic parameters, as well as on response assessment. Data from the literature report on the use of [18F]FDG PET/CT at the baseline with two scans performed before treatment started focused on the time of decision (TD) PET/CT and time of transfusion (TT) PET/CT. Metabolic tumor burden is the most studied parameter associated with disease progression and overall survival, making us able to predict the occurrence of adverse effects. Instead, for post-therapy evaluation, 1 month (M1) PET/CT seems the preferable time slot for response assessment and in this setting, the Deauville 5-point scale (DS), volumetric analyses, SUVmax, and its variation between different time points (∆SUVmax) have been evaluated, confirming the usefulness of M1 PET/CT, especially in the case of pseudoprogression. Additionally, an emerging role of PET/CT brain scans is reported for the evaluation of neurotoxicity related to CAR-T therapies. Overall, PET/CT results to be an accurate method in all phases of CAR-T treatment, with particular interest in assessing treatment response. Moreover, PET parameters have been reported to be reliable predictors of outcome and severe toxicity.Chimeric antigen receptor–engineered (CAR) T cells are emerging powerful therapies for patients with refractory/relapsed B-cell lymphomas. [ Chimeric antigen receptor–engineered (CAR) T cells represent a new powerful tool for the treatment of refractory hematologic malignancies. The therapy is based on the genetic modification of patient-derived T cells by means of viral vectors enabling artificial chimeric antigen receptors expression to recognize specific tumor-associated antigens. The antigen CD19 is widely expressed on B-cell malignancies including non-Hodgkin lymphomas (NHL) such as diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), and follicular lymphoma (FL), thus prompting the use of CAR-T cells targeting CD19 for the treatment of NHL Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography, i.e., [18F]FDG PET/CT, has maintained its importance, although the number of publications specifically dedicated to lymphoma imaging for CAR-T is limited [18F]FDG PET/CT use during CAR-T cell therapy in B-cell lymphomas.As for other lines of treatment in lymphomas, imaging attains an essential role for the evaluation of the patients before and during CAR-T cell therapy. In this context, [ limited . In the 18F]FDG PET/CT imaging contributes significantly both to disease staging, in association with clinical, morphological, and histological data, and for the response assessment of lymphoma patients, thus the imaging is becoming an important part of successful treatment management strategies [[rategies . With th18F]FDG PET/CT at four relevant timings , SUVmax, and its variation between different time points (∆SUVmax), as well as volumetric analyses. The DS is an international scale to visually compare the uptake of lymphoma lesions with referring tissues, such as the liver and the mediastinum. At the time of the response assessment, an uptake greater than that of the liver is usually used as the cut-off for the definition of an unfavorable response . SUVmax To differentiate the responders from non-responders, Cohen et al.  proposedThe quantitative parameters from PET/CT in patients treated with CAR-T cells have been also evaluated by Shah et al.  in sevenDespite the small cohort, they demonstrated that the M1 PET/CT assessment may distinguish responders from non-responders and specifically long-term responders.In fact, long-term responders showed CR at M1 PET/CT, related to the rapid and robust onset of the response. On the contrary, M1 PET/CT clearly showed residual involved sites in PR or signs of early progression in non-responders.In this setting, M1 evaluation seems the preferable time slot for response assessment and may be used to trigger a possible therapeutic intervention.The importance of adequate disease control before the CAR-T has been recently shown by Bailly et al.  in R/R N18F]FDG PET/CT scans in predicting the outcome and thus assisting in the selection of patients benefiting from the treatment. This is important because a consensus statement to guide the management and treatment of patients relapsing after CD19 CART cell therapy has not yet been achieved. Byrne et al. differentiated patients with an early relapse who maintain CD19+ that could beneficiate from a PD-1 inhibitor, from patients with a late relapse which can be considered for other salvage modalities, including reinfusion of the CAR-T cells, ibrutinib, immunomodulatory therapy, or, if the tumor is localized, radiation therapy FDG PET/CT can be challenging, since it can be difficult to distinguish the tumor sites of inflammation from the disease progression, which consequently leads to false-positive findings.The assessment of the response to CAR-T cell therapy with FDG PET/CT, in pseudoprogressive patients there was a partial resolution of the previous FDG-avid lesions consistent with a PR or no-FDG avid residual mass compatible with the complete response (CR) FDG PET/CT imaging, all the involved sites demonstrated an increase in both FDG-uptake (SUVmax 22.8 versus SUVmax baseline 14.5) and size, with additional new FDG-avid findings. To differentiate between progressive metabolic disease and pseudoprogression, a cervical lymph node biopsy was performed with the histological report of ‘’Inflammatory changes without evidence of lymphoma’’. Forty-two days post-infusion, the imaging findings disappeared or improved (∆SUVmax > 66%), indicating a complete metabolic response and supporting the hypothesis of pseudoprogression.The phenomenon of “pseudoprogression” was confirmed by Cohen et al.  also in Of note, the pseudoprogression reported during the CAR-T therapy was more rapid, both in onset and duration, compared with the conventional checkpoint inhibitors. This phenomenon could be an indirect sign of a greater efficacy of CAR-T cell therapy.At the same time, it is fundamental to predict life-threatening consequences produced by an enlargement and local compression in the staging evaluation. Therefore, PET/CT might be useful in the identification of potentially dangerous sites of involvement and estimate PET-parameters to take precautions in preventing damages in local organs .Adverse effects are associated with all cancer therapies, and CAR-T cells are no exception. In cytotoxic chemotherapy, adverse effects are off-target with the permanent genetic modifications of cells in the entire organism and long-term clinically significant consequences; in CAR-T cells infusion, the major toxic manifestations are on-target effects and depend on the specificity of antibody single-chain variable fragments and T-cell activation. These toxic effects are thus reversible when the target cell is removed, or the engraftment of the CAR-T cells is broken off ,64.Primarily, cytokine release syndrome (CRS), neurologic toxicity, and coagulation disorders are all serious limitations in the improvement of CAR-T, particularly in adult ALL patients with more severe manifestations in comparison with patients with other B cell malignancies .The onset of adverse effects is a challenge in CAR-T treatment and the evaluation of baseline PET/CT could have a prominent role through the use of different parameters. Derlin et al.  found th18F]FDG PET/CT imaging in their occurrence.The following paragraphs will undergo more in details on the specific adverse events during CAR-T cell therapy and the role of FDG PET/CT parameters, already at the baseline, have the capability to predict the occurrence of CRS. In the multicenter studies investigating CAR-T for R/R NHL  [18F]FDG PET/CT parameters and CRS, mainly due to the relatively limited cohort of patients and the number of high-grade toxicities.According to our knowledge, there are no cases of patients who have performed PET/CT during CRS. The choice to carry out the follow-up PET after 1 month of infusion has influenced this aspect, since it did not confound findings with the development of CRS, without false-positive results due to inflammation . HoweverANSCEND) ,15,16, tANSCEND) , who assANSCEND)  and WangANSCEND)  and, in ANSCEND)  and ZhouANSCEND)  observedThe neurotoxicity associated with CAR-T cell therapy remains idiosyncratic and poorly characterized. This consists of a variable spectrum of neurophysiological dysfunction arising in CAR-T cells after 1 week of infusion. The most frequent clinical manifestations are encephalopathy, a confusional state, delirium, or changes in mental-status up to coma ,69. ThisIn a patient with suspected ICANS, the first step consists of excluding mimics or other conditions such as infection, cerebral involvement of B cell malignancy, drug toxicity, metabolic derangement, or organ dysfunction. Morphological brain imaging such as computed tomography (CT) and magnetic resonance imaging (MRI) are mostly normal in mild ICANS and even in more severe ICANS .18F]FDG PET/CT may play a prominent role also in the detection of this syndrome, exploiting the evaluation of metabolic activity in the brain, as already carried out in other conditions of metabolic distress and neuronal injuries FDG PET/CT as areas of hypometabolism, or as patterns of elevated velocities on a transcranial Doppler ultrasound. This finding was demonstrated by Rubin et al. [18F]FDG PET/CT. Moreover, in three of these patients, a transcranial Doppler ultrasound proved an elevated flow velocities correlated with the regions of PET hypometabolism. These findings did not correlate with structural lesions and remained without elucidation of possible etiology.Even in sites where MR imaging showed no alteration, focal deficits have been detected with FDG PET/CT of the brain showed a global hypometabolism, especially involving the left hemisphere and the frontal regions with the clinical manifestation of the ideo-motor slowing, nominal aphasia, slowing fluency, frontal release signs, postural myoclonic tremor, and paligraphia. On the other hand, despite the diffuse slowing as depicted on the EEG, the MRI showed the absence of pathological involvement. After intravenous steroid administration, the patient responded completely.Other few studies have mentioned the absent correlation between functional and morphological imaging. Recently, Paccagnella et al.  describe18F]FDG PET/CT brain scan was performed showing a bilateral and diffuse hypometabolism, predominantly in the parietal and temporal cortex, excluding the limbic and cerebellar lobes. Four months after CAR-T cell infusion, a new brain PET/CT showed only prefrontal and temporal hypometabolism. As Vernier FDG PET/CT make it the most valuable imaging tool in CAR-T patients. It turned out to be an accurate method for assessing the extent of the lymphoproliferative process, resulting also in the selection of this treatment. Moreover, at the baseline, the PET parameters are predictive of the outcome and may identify a subgroup of patients mostly benefiting from CAR-T infusion from those tending to have a poorer prognosis and thus less therapeutic benefit. Due to its functional character, M1 PET/CT, and consequently PET parameters, play a central role in response assessment and differential diagnosis between progression and “pseudoprogression”. Last but not least, [18F]FDG PET/CT appears to show promise in predicting the risk of severe toxicity with recent growing interest of PET/CT brain scans in the evaluation of ICANS.The unique features of [


With the steadily increasing abundance of omics data produced all over the world under vastly different experimental conditions residing in public databases, a crucial step in many data-driven bioinformatics applications is that of data integration. The challenge of batch-effect removal for entire databases lies in the large number of batches and biological variation, which can result in design matrix singularity. This problem can currently not be solved satisfactorily by any common batch-correction algorithm.reComBat, a regularized version of the empirical Bayes method to overcome this limitation and benchmark it against popular approaches for the harmonization of public gene-expression data (both microarray and bulkRNAsq) of the human opportunistic pathogen Pseudomonas aeruginosa. Batch-effects are successfully mitigated while biologically meaningful gene-expression variation is retained. reComBat fills the gap in batch-correction approaches applicable to large-scale, public omics databases and opens up new avenues for data-driven analysis of complex biological processes beyond the scope of a single study.We present https://github.com/BorgwardtLab/reComBat, all data and evaluation code can be found at https://github.com/BorgwardtLab/batchCorrectionPublicData.The code is available at Bioinformatics Advances online. Data-driven computational biology greatly depends on the availability of large, integrated datasets to provide the necessary variety and statistical power for state-of-the-art (SOTA) machine and deep learning, as recently demonstrated by Alpha-Fold  . PA is a Gram-negative bacterium with a large genome  in Section 3 as an extension of the ComBat algorithm to handle highly correlated covariates. In the second part of Section 3, we address the issue of assessing the efficacy of the batch-correction by introducing a large variety of evaluation metrics (contribution ii). In Section 4, we benchmark reComBat against a selection of SOTA batch-correction methods on simulated and real-world data. Finally, we present a large, harmonized dataset of PA expression profiles in response to different microenvironmental cues (contribution iii). We conclude Section 4 by demonstrating, as a proof of concept, the biological validity of the harmonized dataset. Section 5 comprises of a discussion and outlook.The article is organized as follows. After reviewing relevant literature in Section 2, we introduce our A variety of batch-correction methods has previously been suggested for bulk and single-cell sequencing data  and score run time, LDA score, Shannon entropy and cluster purity as a function thereof w.r.t. the ground truth. Additionally, data for 887  is beneficial. In the following, we present results only for ridge-reComBat.A hyperparameter screen to optimize regularization strength and type on the default simulated, microarray and bulkRNAsq data yielded best results when ridge regression was used . Notably, if scGen is trained with Zero-Hop labels its performance is greatly improved, however, also prone to overfitting. Overfitting was observed as positive LDA score differences for this method, indicating that a better LDA accuracy was obtained by scGen than possible based on the ground truth data. We only observe degradation of reComBat performance for smaller datasets of 100 samples (given 10 Zero-Hops). Run time was generally very quick and favorable for reComBat compared to Harmony, or scGen (trained on GPU).We benchmark reComBat is successful in removing substantial batch-effects while retaining biologically meaningful signal in real-world data, too. reComBat corrected microarray data colored by batch, Zero-Hops and microbial strain. Uncorrected data clusters by batch, indicating the presence of batch-effects, whereas clustering by biologically meaningful variation (e.g. by strain or Zero-Hop) is observed after correction. Additional overviews of t-SNE embeddings of batch-corrected expression data for all baseline models and data, colored by all design matrix elements are provided in We show quantitatively and qualitatively that reComBat model based on all evaluation metrics  effect when compared to the raw data, independent of the underlying metric and dataset. Despite, markedly different results compared to the uncorrected baseline, Harmony could not achieve sufficient batch-correction characterized by poor performance in classifier and cluster-based metrics throughout. We suggest that the large number of design matrix elements and comparably strong batch-effect could lead to this result. Importantly, reComBat achieved good scores throughout all evaluation metrics for all datasets , Harmony, marker gene and PC elimination to benchmark our linear empirical Bayes method.Public databases play an increasingly important role for data-driven meta-analysis in computational biology. Despite great efforts to harmonize data collection, considerable, yet unavoidable, biological/technical variation may mask true signal if data are pooled from several sources. To draw generalizable conclusions from agglomerated data, it is essential to correct such batch-effects in a setting where overlapping samples, or standardized controls, are unavailable. When large numbers of (>20) batches coincide with desired biological variation, a range of standard batch-correction algorithms are inapplicable. We would like to stress that this evaluation scenario greatly differs from previously analyzed batch-correction settings where comparably few (2–5) batches with large number of overlapping samples were included, or comparably small batch-effects within a single study were corrected  was given to either of the methods. We demonstrate not only the superiority of reComBat compared to these baselines but, by providing a large variety of evaluation metrics, also give a notion of overall performance.In case of a singular design matrix reComBat. Due to this fact, we excluded some popular deep models [e.g. normAE  our method is readily available to the community. We also make the harmonized datasets with their metadata available to the wider research community (https://github.com/BorgwardtLab/batchCorrectionPublicData).With >5000 citations, ComBat is one of the most popular batch-correction methods today applied to a large variety of data types and organisms (We have addressed the challenge of harmonizing large, and highly diverse public data for downstream meta-analysis. Aiming at high community acceptance and a computationally efficient solution, we extend the well-established ComBat algorithm through the addition of regularization. We evaluate our novel algorithm on simulated, and public microarray and bulkRNAsq data. A variety of evaluation metrics attest comparable, or superior correction of batch-effects as established baseline models. Our analysis constitutes a proof of principle to motivate and enable further large-scale meta-analyses.vbac071_Supplementary_DataClick here for additional data file.


Tendon injuries are one of the most common musculoskeletal disorders for which patients seek medical aid, reducing not only the quality of life of the patient but also imposing a significant economic burden on society. The administration of growth factors at the wound site is a feasible solution for enhancing tendon healing. Platelet-derived growth factor-BB (PDGF-BB) has a well-defined safety profile compared to other growth factors and has been approved by the Food and Drug Administration (FDA). The purpose of this review is to summarize the role of PDGF-BB in tendon healing through a comprehensive review of the published literature. Experimental studies suggest that PDGF-BB has a positive effect on tendon healing by enhancing inflammatory responses, speeding up angiogenesis, stimulating tendon cell proliferation, increasing collagen synthesis and increasing the biomechanics of the repaired tendon. PDGF-BB is regarded as a promising candidate in tendon healing. However, in order to realize its full potential, we still need to carefully consider and study key issues such as dose and application time in the future, so as to explore further applications of PDGF-BB in the tendon healing process. Musculoskeletal disorders are characterized by high morbidity and disability in the epidemiology of many countries and are therefore an important branch of public health . Tendon-+ cells in Achilles tendon healing. They concluded that the reduction in the amount of α-SMA protein in the presence of PDGF-BB could be judged as a positive, anti-fibrotic outcome, which was thought to result in less scar formation during the healing process [Some in vitro studies have shown that different growth factors including fibroblast growth factor-2 (FGF-2), transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) may play a key role in regulating angiogenesis, matrix formation and cell proliferation in tendons, with the goal of improving tendon healing . Further process ,12,13. M process . On the PDGF-BB, as the most studied heterodimer in the PDGF growth factor family, is the only dimer that can bind to three different surface platelet-derived growth factor receptors (PDGFRs) and trigger different signal pathways. Its role in tendon healing is multifaceted. Once PDGF-BB combines with three different surface PDGFRs, it will initiate the PDGF signal cascade, in which different cellular processes are affected through different signal pathways ,15,16.Several studies have demonstrated the role of PDGF-BB in promoting tendon healing. Nevertheless, the elaborate mechanisms have not been fully elucidated. Hence, the purpose of this review is to summarize the role and mechanism of PDGF-BB in tendon healing, to further explore the methods of PDGF-BB in promoting tendon healing in the future and to provide a theoretical basis for follow-up researchers.Search strategy. (i) Search site: Articles are from PubMed, a database of papers on biomedical science. (ii) Database: MEDLINE. (iii) Keywords: platelet-derived growth factor-BB, PDGF-BB, tendon injury, tendinopathy, tendon healing. (iv) Boolean algorithm: (“platelet-derived growth factor-BB” OR “PDGF-BB”) AND . (v) Retrieval timeframe: We searched the selected journals published from 1982 to 2022. (vi) Inclusion and exclusion criteria: Articles were included if the topic was related to platelet-derived growth factor-BB and tendon healing, and the article type was a review or an experimental paper. The search process is shown in A number of experimental studies have shown that PDGF-BB has an important role in the process of tendon healing ,11,13.Yoshikawa, Thomopoulos and Haupt et al. all investigated the benefits of PDGF-BB in tendon healing through in vitro experiments. Yoshikawa et al. investigated the stimulatory effects of PDGF-BB on cell proliferation and matrix synthesis in rabbit tendons. They found that PDGF-BB stimulated matrix synthesis and cell proliferation in rabbit tendons in a dose-dependent manner in vitro, supporting the possibility of the clinical application of PDGF-BB for enhancing intrinsic tendon metabolism . ThomopoOn the other hand, many in vivo experiments have demonstrated the important role of PDGF-BB in tendon healing. Hildebrand et al. investigated the effects of PDGF-BB application on mature male New Zealand White rabbits and then found that the application of PDGF-BB to the ruptured right medial collateral ligament dramatically improved the failure absorption energy, ultimate load and ultimate elongation values of the femoral–MCL–tibial complex compared to the no growth factor group. Furthermore, higher doses of PDGF-BB were able to better refine the structural properties of the femoral–MCL–tibial complex compared to lower doses of PDGF-BB. Simultaneously, the PDGF-BB alone treatment group produced better results compared with a combination of PDGF-BB plus TGF-β1. In this study, the ability of PDGF-BB to improve healing in the early stages of ligament repair has been confirmed .Furthermore, Kovacevic et al. set up a rat rotator cuff repair model and randomly divided the rats into five groups: the repair-only group, collagen scaffold-only group and three collagen scaffold groups with different doses of PDGF-BB. The results of the experiment showed that after 5 days, delivery of rhPDGF-BB on the scaffold showed a dose-dependent response in terms of cell angiogenesis and proliferation compared to the repair-only and scaffold-only groups. This result also confirms that the delivery of rhPDGF-BB on collagen scaffolds strengthens angiogenesis and cell proliferation in the early stages of healing. At the same time, this study illustrates the importance of ongoing understanding and optimization of growth factor delivery systems that can lead to an enhanced healing environment to improve structural integrity and reduce re-injury rates after tendon healing .Unlike others, Younesi et al. applied PDGF-BB growth factor solution to heparinized electrochemically aligned collagen sutures (ELAS), allowed the solution to be completely absorbed by the otherwise dry sutures and then used it to suture the completely torn right third toe flexor muscle of mature White Leghorn chickens. The results showed that the combination of heparinized ELAS with PDGF-BB improved biomechanics and vascularity during tendon healing 12 weeks after primary repair .® (DP) (polyester urethane), conveying PDGF-BB, as an implant to improve tendon healing. In vivo evaluation after three weeks showed positive results, with the delivery of PDGF-BB via bioactive DP tubing leading to a 2-fold increase in the tensile strength on treated tendons, with no additional pro-fibrotic effects, thus providing a prospective bioactive implant for clinical applications in the field of tendon healing [Ultimately, Evrova et al. established a rabbit Achilles tendon complete tear model and then used electrospun tubes made of DegraPol healing .In conclusion, these studies suggest that PDGF-BB has a positive effect on tendon healing by promoting angiogenesis, cell proliferation and collagen production as well as increasing the biomechanics of the repaired tendon. The biological responses embodied by PDGF-BB in different animal experiments are shown in PDGF-BB exerts its cellular action by binding to the receptor and entering the cell. LIANG et al. mentioned that increasing PDGF-BB concentration was ineffective in facilitating cell proliferation when the number of its receptors was limited and they were all occupied . PDGF-BBAlthough the heterodimeric receptor complexes of PDGF may have distinctive properties, most of the information on the function and role of homodimers is accessible. Both α and β receptor homodimers transmit very effective mitogenic signals, but their effects on the actin filament system are distinct. β receptors can mediate the formation of circular actin structures on the dorsal surface of cells, whereas α receptors cannot . FurtherAmong the events that promote tendon healing, the administration of growth factors at the wound site is a viable strategy. After tendon injury, PDGF-BB is secreted by platelets along with other growth factors. When PDGF-BB is applied as a potent chemokine, it can accelerate tendon healing by promoting mitosis and angiogenesis. As a universal heterodimer of PDGF, PDGF-BB initiates a signaling cascade once it combines with its receptor, and different signaling pathways affect different cellular processes . It shouDifferent molecular elements and mechanisms are present in the different phases of tendon healing. The inflammatory phase tends to occur within the first hour after tendon injury; immediately after injury, platelets and inflammatory cells (such as macrophages and neutrophils) release a mixture of cytokines and growth factors that are attracted to the wound site and produce tumor necrosis factor (TNF) or growth factors involved in neovascularization, such as PDGF, fibroblast growth factor (FGF), VEGF and so on, as shown in The inflammatory phase, as the first stage of tendon healing, is an important driver for normal tissue healing . While cSuccessful tissue healing requires an initial inflammatory response . A previThe series of inflammatory responses to harmful stimuli is considered an essential physiological process in the body . HoweverDue to the characteristics of connective tissue, tendons have a weak vascular network, which makes tendon healing a long-term process; therefore, the angiogenic response is a key event in the tendon healing process. This is a complex multistep process . StudiesThe PI3K signaling pathway is one of the major signaling pathways induced by the activation of PDGF-BB and contributes to many cellular processes, including angiogenesis as well as cell proliferation, survival and motility ,44. IntePDGF-BB, as a growth factor known to enhance mitogenesis, can promote angiogenesis by directly regulating the proliferation of vascular endothelial cells and inducing cell migration via PDGFR-β ,52. GeneApart from that, PDGF-BB has also been proved to stimulate angiogenesis by inducing erythropoietin (EPO) production in stromal cells . ExperimEventually, angiogenesis mediated by the regulation of specific integrins by PDGF-BB may be an important parameter for successful tendon repair . AngiogeTendon cells are specialized fibroblasts. PDGF-BB has been demonstrated to induce the mitosis and proliferation of fibroblasts. Several experimental studies have shown that PDGF-BB can stimulate the proliferation of tendon cells and, similarly, has a positive stimulatory effect on collagen synthesis in tendon cells.Above all, it has been shown that PDGF-BB can be involved in fibroblast proliferation, transformation and collagen synthesis by activating the PI3K/AKT signaling pathway . PreviouMoreover, PDGF-BB is capable of enhancing α1β1 integrin-mediated cellular collagen matrix remodeling by elevating extracellular signal-regulated kinase (ERK)/activator protein-1 (AP-1) activity. Integrin-mediated signaling has previously been shown to influence cellular behavior together with growth factors, such as migration, proliferation, and survival, among others, while the importance of specific integrin regulation for intra-tendon healing has also been reported ,70,71. TAdditionally, it has been suggested that PDGF-BB does not directly affect collagen synthesis. It acts as a potent chelator of wound macrophages and fibroblasts, possibly by inducing an increase in endogenous TGFβ1, which in turn stimulates new collagen synthesis and enhances wound healing ,74.As we all know, mitosis is one of the ways in which an organism’s cells proliferate, and mitotic activity can be used as a measure of the cells’ proliferative potential. PDGF-BB was found to be the most efficacious mitogen of all isoforms of PDGF and enhances mitotic activity in a concentration-dependent manner in the range from 1.0 to 50.0 ng/mL . Both thPDGF-BB acts as a crucial regulator of cellular function as it can promote tendon healing by inducing cell differentiation ,80. Src Tendon healing is a complex and lengthy process that involves three overlapping phases. The administration of growth factors is a viable strategy in the event of promoting tendon healing. As a growth factor that accelerates mitogenesis, PDGF-BB has been shown to support tendon healing . PDGF-BBTendon cell proliferation is one of the main responses by adding PDGF-BB in a dose-dependent manner or in complete or serum-free medium . MeanwhiMoreover, it has been shown that PDGF-BB can induce the differentiation of ASCs to tendon cells, which facilitates the regeneration of tendon tissue . HoweverIn recent years, many studies have continuously optimized growth factor delivery strategies, especially PDGF-BB, and developed novel drug delivery devices to control the release of growth factors in time and space to reduce the rapid removal of growth factors at the wound site and then achieve a positive outcome of fostering healing ,11,15,89


Ovarian cancer (OvCa) is one of the most widespread malignant tumors, which has the highest morbidity and unsatisfactory clinical outcomes among all gynecological malignancies in the world. Previous studies found that cancer-associated fibroblasts (CAFs) play significant roles in tumor growth, progression, and chemoresistance. In the current research, weighted gene co-expression network analysis (WGCNA), univariable COX regression, and the least absolute shrinkage and selection operator (LASSO) analysis were applied to recognize CAF-specific genes. After multiple bioinformatic analyses, four genes  were identified as OvCa-specific CAF markers and used to construct the prognostic signature (CAFRS). Furthermore, the specificity of the four genes' expression was further validated at the single-cell level, which was high-selectively expressed in CAFs. In addition, our results showed that CAFRS is an independent significant risk factor affecting the clinical outcomes of OvCa patients. Meanwhile, patients with higher CAFRS were more likely to establish chemoresistance to platinum. Besides, the CAFRS were notably correlated with well-known signal pathways that were related to tumor progression. In summary, our study identifies four CAF-specific genes and constructs a novel prognostic signature, which may provide more insights into precise prognostic assessment in OvCa. Due to ll level . In addill level . CollectIncreasing numbers of evidence suggested that recognized markers, such as α-smooth muscle actin and fibroblast activation protein α, are carried on CAFs . HoweverConsidering the advantages and limitations of single-cell sequencing and RNA sequencing or microarray technology, we firstly assessed the infiltration status of the stroma and CAFs using published algorithms that assess the abundance or levels of infiltration cell populations according to the expression values of some well-known signatures. Then, specific CAF genes were identified using weighted co-expression network analysis (WGCNA) by linking the fraction of CAFs with the relative gene module, and the clinical value of the gene signature was further explored and verified in many other microarray datasets. Finally, the expression specificity of gene signatures of OvCa CAFs was further verified in a single-cell transcriptome dataset.https://www.ncbi.nlm.nih.gov/geo/) and UCSC Xena website (https://xenabrowser.net/datapages/) were used to obtain gene expression profiles of OvCa patients. After screening, six OvCa-related datasets, including GSE9891 (The Gene Expression Omnibus (GEO) portal  method  was usedPrevious studies identified shared gene signatures and biological mechanisms in type 2 diabetes and pancreatic cancer by utilizing the WGCNA method . To furtp-value < 0.05). Subsequently, the least absolute shrinkage and selection operator (LASSO) regression algorithm was utilized to screen the potential prognostic genes, which were defined as CAF-specific markers. The risk score of the prognostic model based on CAF-specific markers (CAFRS) of patients was calculated based on the linear combination of the expression values of CAF-specific markers multiplied by the corresponding LASSO coefficients. To confirm the role of the risk score in the prediction of prognosis, the OvCa patients were divided into the high- and low-CAFRS groups according to the 50% cutoff of the CAFRS.Next, CAF-specific markers were selected to construct the gene risk score following the criteria. Firstly, genes with GS ≥ 0.5 and MM ≥ 0.5 in the module which had the highest correlation with CAF fraction were selected for further analysis. Then, the univariable COX regression analysis was conducted to extract genes significantly associated with OS  and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed by using the R package clusterProfile. The top ten enriched GO and KEGG pathways with the most significant http://satijalab.org/seurat/) R toolkit .In order to minimize the technical batch effects among individuals and experiments, we used the “RunHarmony” function in R package harmony  to integp-value < 0.05 was deemed to be statistically significant, and labeled with *p-value < 0.05, **p-value ≤ 0.01, ***p-value ≤ 0.001, and ****p-value ≤ 0.0001.All statistical analyses were handled using R-4.0.4. The significant difference in continuous variables between the two groups was assessed using the Wilcoxon rank-sum test, while categorical variables were compared by the chi-square test. Prognostic values were evaluated using the log-rank test. For all analyses, a two-paired p = 0.03, Hazard Ratio [HR] = 1.6) and Stromal Score  were remarkably related to poor OS  was selected as the soft threshold to ensure a scale-free network (p < 0.001) and levels of stromal cells . The GS and MM values for the yellow module in CAF and StromalScore were displayed in scatter plots . The univariable COX regression analysis results showed that the improve of CAFRS  and stage  were risk factors for OS (p < 0.001) and stage  were independent prognostic factors in OvCa  prognostic signature was developed to predict the clinical outcomes and the response to anti-tumor therapies in gastric cancer . Howeveret al. proved that the curative effects of immune checkpoint blockade could be reduced by extracellular matrix (Furthermore, we also found that OvCa patients with high CAFRS were more likely to exhibit resistance to platinum. And the CAFRS were notably correlated with well-known signaling pathways related to tumor progression. Several pieces of evidence reported that CAFs are associated with therapeutic resistance . The cror matrix . Due to r matrix .Although our research recognized and confirmed the clinical significance of OvCa CAF-specific markers and the risk score constructed based on them, further clinical studies are warranted to validate that these signatures can be potential biomarkers or targets of CAFs in OvCa. In addition, given the connections between CFAs and TME, whether the CAF-specific signature could predict the response to immune checkpoint blockade is also worth exploring.To sum up, based on multiple bioinformatics analyses, we reported that the enhanced infiltration of fibroblast in OvCa was remarkably associated with worse clinical outcomes. In addition, the 4-gene signature with prognostic significance, which was identified by WGCNA and verified in other independent cohorts and single-cell RNA-sequencing datasets, was significant for future studies on CAFs and shed novel insights into CAFs-target therapy in OvCa.


Gliomas are the most common malignant tumor in the brain. As with other tumors, the progression of glioma depends on intra-tumoral angiogenesis. However, the effect of angiogenesis on gliomas is still not fully understood. In this study, we developed an angiogenesis pathway score using Gene Set Variation Analysis (GSAV) in R to assess the status of intra-glioma angiogenesis in The Cancer Genome Atlas (TCGA), Chinese Glioma Genome Atlas , and GSE16011 datasets. We found that the angiogenesis pathway score not only accurately predicted the prognosis of glioma patients, but also accurately distinguished the malignant phenotype and immune characteristics of gliomas. In addition, as an independent prognostic factor, the score could predict glioma sensitivity to radiotherapy and chemotherapy. In summary, we used the angiogenesis pathway score to reveal the relationship between glioma angiogenesis and the malignant phenotype, immune characteristics, and prognosis of glioma. As the most frequent malignant primary intracranial tumor, gliomas account for approximately 80% of brain tumors and can be further divided into glioblastoma (GBM) and low-grade gliomas (LGG) based on histology and molecular features . AlthougThe pathophysiologic mechanisms underlying angiogenesis have attracted much attention in research due to its vital role in wound repair and cancer pathogenesis. In cancer, the outgrowth of capillary sprouts is more prominent and continuous than that in inflammation or in fresh wounds, which indicates that tumor progression partially relies on angiogenesis . CompareTumor angiogenesis is regulated by many factors including vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs), fibroblast growth factors (FGFs) and their receptors, angiopoietin (ANGPT), tyrosine kinase with immunoglobulin-like and EGF-like domains (TIE) signaling, and also transforming growth factor-betas (TGF-βs). They play a vital role in angiogenesis in various types of tumors . In addiIn vivo and in vitro experiments indicate that adhesion of leukocytes to tumor vascular endothelial cells is significantly reduced, which in turn leads to changes in the immune cell composition within the tumor microenvironment [Angiogenesis affects tumor cells and their microenvironment. Blood vessels provide tumor cells with oxygen and nutrients. They also supply a route for tumor cells to metastasize which promotes the malignant phenotype of tumors . In breaironment , 18.Angiogenesis also has an important influence on glioma malignant phenotype, including cancer cell invasion, stem cell phenotype, genetic instability, altered metabolism, epithelial to mesenchymal transition (EMT), and anti-tumor immunosuppression –21. It iIn this study, we used Gene Set Variation Analysis (GSVA) score to define the “HALLMARKER_ANGIOGENESIS” gene set score. The angiogenesis pathway score was associated with the state of angiogenesis in gliomas and also accurately distinguished the malignant phenotypes and immune characteristics of gliomas. In addition, the angiogenesis pathway score independently predicted prognosis and glioma sensitivity to treatment.The GSVA score of the “HALLMARK_ANGIOGENESIS” gene set retrieved from MSigDb was determined by using the global glioma gene expression data and used as angiogenesis pathway score. The median value of the angiogenesis pathway score divided a given cohort into high- and low-score groups. To explore whether the angiogenesis pathway score could reflect the level of intra-tumoral angiogenesis, we compared the expression of angiogenesis-related genes in the high- and low-score groups in the TCGA, CGGA, and CGGA (array) datasets. As shown in In contrast to vessels in nonneoplastic tissue, tumor vasculature is highly chaotic often showing dilation and tortuous growth patterns . This maIn addition to being regulated by glioma cells, angiogenesis is also regulated by the environment. Hypoxia is an important environmental factor that regulates angiogenesis . Thus, wIn TCGA dataset, KM analysis showed that glioma patients in the low-score group displayed significantly longer OS than that seen in the high-score group . In addiIDH had a significantly higher angiogenesis pathway score than those with mutant IDH  datasets, as the grade of glioma increased, the angiogenesis pathway score increased significantly . In additant IDH . These aMicrovessels are an important component of the glioma microenvironment and increased angiogenesis in gliomas improves microcirculation. Bearing this in mind, we speculated the angiogenesis pathway score might be associated with the purity of glioma and the enrichment degree of immune cells within the glioma microenvironment. To examine this, we first checked correlation between the angiogenesis pathway score and glioma purity. In the TCGA, CGGA, and CGGA (array) datasets, the stromal, immune, and ESTIMATE scores were significantly positively correlated with the angiogenesis pathway score. However, the correlation between tumor purity and the angiogenesis pathway score was just the opposite . Additio+ T cells, play an important role in the immune clearance of tumor cells, however according to our data, the high enrichment of immune cells was related to a poorer prognosis. We speculated that angiogenesis may be related to immune tolerance of gliomas. Immune checkpoints play a vital role in the maintenance of self-tolerance under normal physiological conditions and is dysregulated by tumors as a crucial factor in immune resistance [Many immune cells, such as CD8sistance . Thus, wsistance –36. The To further explore the significance of the angiogenesis pathway score in glioma, we conducted a GSEA analysis of the Hallmark gene sets in the high- and low-score groups. Immune response-related gene sets, such as IL2-STAT5 signaling, interferon (IFN)-α and IFN-γ responses, were significantly enriched in the high-score glioma group in TCGA, CGGA, and CGGA (array) datasets , which cFurthermore, we also found that the enrichment levels of epithelial mesenchymal transition (EMT) and NOTCH signaling in the high-score group were significantly higher than those in the low-score group in the TCGA, CGGA, and CGGA (array) datasets . FurtherDespite compelling effects in other cancers, the outcomes of anti-angiogenic therapies, such as bevacizumab, and cilengitide, were disappointing in gliomas . GliomasSince the angiogenesis pathway score has such a powerful potential in predicting the prognosis of glioma, we believed it may be an independent prognostic factor for glioma. To confirm this conjecture, univariate and multivariate Cox regression analyses were conducted in the TCGA, CGGA, and CGGA (array) databases to analyze the independence of angiogenesis pathway score in the prognosis of glioma. As shown in In this study, we explored the angiogenesis pathway score, which was measured by GSVA score based on the “HALLMARK_ ANGIOGENESIS” gene set retrieved from MSigDb, in TCGA, CGGA, CGGA (array), and GSE16011 datasets. This was done to reveal the relationship between intra-tumoral angiogenesis in glioma and features such as cancer aggressiveness, immune microenvironment, metastasis, and treatment response. To the best of our knowledge, utilizing GSAV score to explore the clinical relevance of intra-tumoral angiogenesis in glioma has not been previously reported.The angiogenesis pathway score correlated with significantly enriched expression for VEGF-related genes, vascular endothelial cell markers, vascular stability related- genes and hypoxia-related genes. This indicates the score can accurately assess the status of intra-tumoral angiogenesis in glioma. The prognosis of glioma patients with high angiogenesis pathway score was significantly worse than that of low-score patients. High angiogenesis pathway score gliomas demonstrated a predilection for aggressive clinicopathological characteristics, such as higher grade, IDH wildtype genotype, 1p19q non-codeletion, MGMT promoter unmethylated status, and mesenchymal subtype. In addition, high score correlated with lower tumor purity and greater immune cell infiltration, regardless of pro- or anti-cancerous immune cells, and also higher expression of most immune checkpoints. The angiogenesis pathway score decreased as glioma sensitivity to radiotherapy and chemotherapy increased. Furthermore, the angiogenesis pathway score acted as an independent prognostic factor to accurately predict the outcome of glioma patients.The structure of vessels in tumor is dissimilar from that in normal tissue. Blood vessels in tumor branch irregularly, follow a crisscross pattern, are highly permeable, and are often larger with irregular luminal diameter . It is pTumor angiogenesis is not only affected by tumor cells. Other cells in the tumor microenvironment also have a regulatory effect. For instance, tumor-associated macrophages participate in all stages of angiogenesis in gliomas by releasing paracrine factors such as WNT7b and M-CSF . This unIn conclusion, our study revealed that the angiogenesis pathway score, as an independent prognostic factor, can distinguish the malignant phenotype of glioma and accurately predict the immune characteristics and prognosis of glioma.The present study has been reviewed and approved by the Institutional Review Board (IRB) of the Sheng Jing hospital of China Medical University, and all patients have provided their written informed consent. A total of 8 samples were collected and sent for bulk RNA sequencing. RNA sequencing was performed by the Illumina Next Generation Sequencing Service at the Beijing Genomics Institution in Shen Zhen. The diagnosis of glioma in each patient was rendered by integrated evaluation of preoperative imaging and postoperative pathology.http://cancergenome.nih.gov/). The Chinese Glioma Genome Atlas (CGGA) mRNA expression data (mRNAseq_325 and mRNA-array) and relevant clinicopathological information were obtained from the CGGA database (http://www.cgga.org.cn) (CGGA will be used to refer to the CGGA mRNAseq_325 dataset unless otherwise specified). The GSE16011 dataset was retrieved from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo). The background correction and normalization of the GSE16011 raw data was conducted using the robust multi-array analysis (RMA) method of the affy R package. Sample characteristics, including detailed data for each research object, are shown in The Cancer Genome Atlas (TCGA) glioma gene expression profile and corresponding clinical information were retrieved from TCGA database  (v7.2), was employed to calculate the angiogenesis pathway score using the GSVA package in R. Gene Set Enrichment Analysis (GSEA) was conducted using GSEA software (v4.1.0) and the GSEA results with false discovery rate (FDR) <0.25 were considered as statistically significant , 45.The ESTIMATE R package is designed to infer tumor purity and the presence of infiltrating stromal/immune cells within tumor tissues using gene expression data . We usedGSVA package in R can identify the enrichment of immune cells within tumor microenvironment using the gene expression signatures [The single-sample GSEA (ssGSEA) method of gnatures . We usedGlioma samples were divided into high- and low-score groups according to the median angiogenesis pathway score. The overall survival (OS) difference between the high- and low-score groups was determined using the Kaplan-Meier (KM) method of the survival R package. Univariate and multivariate Cox regression analyses were conducted using the survival package to assess the independence of the angiogenesis pathway score in predicting the OS of glioma patients.We calculated the semi-inhibitory concentration (IC50) values of chemotherapeutic drugs using the “pRRophetic” package.U test was applied for multiple groups. The correlation of two variables was accessed by Pearson’s correlation test. P value < 0.05 was considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.The R software (version 4.0.2) and GraphPad Prism (version 7.0.0) were used for most statistical analyses and graphing in this study. The R package “limma” was used to identify the difference of immune cells enrichment scores and the expression of immune check points between high- and low-score groups. Wilcoxon signed-rank test was used to compare gene expression between two groups while Mann-Whitney http://www.cgga.org.cn/ (CGGA), https://portal.gdc.cancer.gov/ (TCGA), and https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE16011 (GSE16011).The datasets used in this study can be downloaded from: Supplementary FiguresSupplementary Table 1


There is uncertainty whether long-term use of proton-pump inhibitors can cause gastric adenocarcinoma (GAC) and oesophageal adenocarcinoma (OAC). This study aimed to determine how discontinuation of long-term PPI therapy influences the risk of GAC and OAC.This population-based cohort study included all long-term users of PPI therapy in Sweden in 2005–2018 was based on Swedish nationwide health registry data. The exposure was discontinuation of long-term PPI therapy, defined as no dispensation of PPI following inclusion and used as a time-varying variable, compared to remaining on PPI. Main outcomes were GAC and OAC, while oesophageal squamous cell carcinoma (OSCC) was included as a comparison outcome. Incidence rate ratios (IRR) with 95% CI adjusted for age, sex, comorbidity, obesity, diabetes, hyperlipidaemia, NSAIDs/aspirin, and statins were calculated with Poisson regression.Among 730,176 long-term PPI users  with 4,210,925 person-years at risk (median 5.5 person-years), 439,390 (60.2%) discontinued PPIs. In total, 495 developed GAC, 598 OAC, and 188 developed OSCC. PPI discontinuation was associated with decreased risk of GAC  and OAC , but not OSCC  compared to continued PPI use. Stratified analyses showed decreased point estimates across most age categories and both sexes for GAC and OAC risk among participants discontinuing PPI therapy.Discontinuation of long-term PPI therapy may decrease the risk of GAC and OAC, suggesting that physicians should consider ceasing prescribing long-term PPI in patients without continued indication for its use.The online version contains supplementary material available at 10.1007/s00535-022-01930-3. The main indications for long-term PPI use are gastro-oesophageal reflux disease (GERD) and gastric ulcer prevention in long-term users of non-steroidal anti-inflammatory drugs (NSAIDs) or aspirin  in a single year. Only long-term users of PPI were included because short-term use is unlikely to be carcinogenic, and 180 tablets have been considered a reasonable cut-off for long-term use in previous publications [The study cohort consisted of all individuals in Sweden using long-term PPI therapy during the study period according to the Prescribed Drug Registry. The definition of long-term PPI therapy was dispensation of ≥ 180 tablets of PPIs [ications , 15. Cumications . ExcludeThe study exposure was discontinuation of long-term PPI therapy, which was defined as the lack of dispensation of PPIs in the years following study inclusion. Participants dispensing any amount of PPI following inclusion were categorized as remaining on PPI therapy. PPI therapy was used as a time-dependent covariate and each individual study participants could therefore contribute person-time to both categories remaining on and discontinuing PPI therapy, which is recommended to avoid immortal time bias  . Person-Gastric adenocarcinoma, oesophageal adenocarcinoma and oesophageal squamous cell carcinoma were defined by a set of diagnosis codes according to the 7th version of the International Classification of Diseases (ICD-7), combined with histology codes signifying adenocarcinoma or squamous cell carcinoma (online Appendix). Person-time after the diagnosis date of any of the three outcome tumors was censored, meaning that each participant could contribute to only one outcome.Helicobacter pylori eradication therapy, NSAIDs or aspirin use, and statin use. All ICD, ATC, and NOMESCO codes used to define the confounders are listed in the online Appendix.Twelve variables were considered potential confounders: age, sex, Charlson Comorbidity Index , tobaccoHelicobacter pylori eradication therapy occurred in a too small frequency of study participants to be included in the regression model, participants exposed to these factors were instead excluded in sensitivity analyses. All variable definitions, categorizations and analyses adhered to a detailed study protocol finalized before initiation of the data management. The analyses were conducted by a senior biostatistician (FM) using the statistical package SAS version 9.4 .Poisson regression was used to calculate incidence rate ratios (IRR) with 95% confidence intervals (CI) as the measure of relative risk comparing discontinuation of long-term PPI therapy with continued long-term PPI use in relation to risk of the cancer outcomes. Follow-up started 1 year after inclusion into the study cohort and ended on the date of any of the three cancer outcomes, death, or end of study period, whichever occurred first. Tumors that developed during the first year following discontinuation or remaining on PPI therapy  were censored to allow for latency . The IRRs were adjusted for age (categorized in approximate quartiles), sex , comorbidity , obesity, hyperlipidaemia or diabetes , NSAID or aspirin use (yes or no), and statin use (yes or no). To assess effect modification, we conducted analyses stratified for age, sex and follow-up time. Because tobacco smoking-related diagnoses, Barrett’s esophagus, anti-reflux surgery, and The cohort included 730,176 long-term users of PPI  Table . Apart fA total of 598 participants developed oesophageal adenocarcinoma, which corresponded to an incidence rate of 14.2 cases per 100,000 person-years. Discontinuation of long-term PPI use was associated with a decreased risk of oesophageal adenocarcinoma compared to continuation of PPI therapy  Table .Table 4IDuring follow-up, 188 cases of oesophageal squamous cell carcinomas were detected, resulting in an incidence rate of 4.5 cases per 100,000 person-years. Discontinuation of long-term PPI therapy did not decrease the risk , and no clear trend was observed in the stratified analyses  person-years. The adjusted IRRs after discontinuing long-term PPI were 1.05 (95% CI 0.80–1.38) for gastric adenocarcinoma, 0.83 (95% CI 0.66–1.04) for oesophageal adenocarcinoma, and 0.93 (95% CI 0.60–1.44) for oesophageal squamous cell carcinoma.The sensitivity analyses excluded 81,881 participants with tobacco smoking-related disorders, Barrett’s esophagus, anti-reflux surgery, or This first study on the topic indicates that discontinuing long-term PPI therapy is associated with a decreased risk of gastric adenocarcinoma and oesophageal adenocarcinoma, and not of oesophageal squamous cell carcinoma.Strengths of the study include the population-based design and large sample size, where virtually all long-term users of prescribed and dispensed PPIs in Sweden were included, which counteracted selection bias and improved statistical power. Defining drug use as dispensations from a registry rather than by questionnaire had the advantages of prospective data collection, thus avoiding misclassification due to issues of recalling, and actual dispensing (and paying for) the prescribed medication, which reduced non-compliance. The use of nationwide complete registries ensured long and complete follow-up for all endpoints. Including oesophageal squamous cell carcinoma as a control outcome with no association with PPI use facilitated the interpretation, and the lack of association between discontinuation of long-term PPI use and this cancer supported the validity of the findings for gastric and oesophageal adenocarcinoma.Helicobacter pylori eradication therapy ensured that virtually all known confounders were accounted for.Using PPI as a time-varying covariate and the use of 1 year latency minimized the risk of immortal time and latency bias . CausaliOther weaknesses of the study include the inability to accurately examine duration and cumulative dosage of PPI use, given the methodological pitfalls and the risk of introducing immortal time using this approach in cohort studies . The inaHelicobacter pylori infection, while smoking is a weak risk factor, and Barrett’s esophagus and anti-reflux surgery are not associated with this tumor. Helicobacter pylori or its treatment is not an indication for long-term PPI use and should therefore not be a major confounder for the association between PPI discontinuation and risk of gastric adenocarcinoma, although some patients with gastroduodenal ulcers may be prescribed PPI indefinitely. Nevertheless, after excluding participants with Helicobacter pylori eradication therapy and tobacco smoking-related disorders in a sensitivity analysis, the association was attenuated to null for gastric adenocarcinoma. Although this sensitivity analysis had low power, the association should be interpreted with some caution.The decreased risk of gastric adenocarcinoma following discontinuation of long-term PPI use was hypothesized and also observed in the main analysis. Among the exclusion variables, the main risk factor for gastric adenocarcinoma is Helicobacter pylori-eradicated participants, found that long-term use of PPIs was associated with an over twofold increased risk of gastric cancer  [Several previous studies have indicated a strong association between PPI use and gastric adenocarcinoma. In this study, we found an incidence of gastric adenocarcinoma in patients remaining PPI in parity with the incidence in the Swedish general population (of which approximately 6% use PPI), but the risk was slightly lower in patients discontinuing PPI . A meta-42–4.20) . A Swedi42–4.20) . The lowHelicobacter pylori can progress in PPI users and promote carcinogenesis, which could be counteracted by PPI discontinuation [Helicobacter pylori were excluded in the present study. While Helicobacter pylori eradication therapy decreases the risk of gastric adenocarcinoma, it should be noted that atrophic gastritis represents a late stage and may still progress to intestinal metaplasia following eradication therapy [Some mechanisms can explain a potentially decreased risk of gastric adenocarcinoma after discontinuation of long-term PPI therapy. First, discontinuation can normalize the elevated gastrin levels associated with long-term PPI use , 26, whiinuation –31. This therapy , 33.Similar to the results of gastric adenocarcinoma, the risk of oesophageal adenocarcinoma was decreased in this study. However, the study population represented a group at clearly higher risk of oesophageal adenocarcinoma compared to the corresponding Swedish general population , likely Other substances present in the refluxate among patients with GERD, particularly bile acids, have been proposed as causative of oesophageal adenocarcinoma. Biliary reflux may be a cause of refractory GERD not responding well to PPI therapy, and most patients with GERD also have pathologically increased biliary reflux . These aThe possible risk reduction of oesophageal and gastric adenocarcinoma in individuals discontinuing long-term PPI observed in this study are not indiscriminately generalized to all patients on long-term PPI therapy. PPIs are well tolerated by most patients and usually offer effective symptom relief for GERD and is essential in the prevention of upper gastrointestinal bleeding among patients on anticoagulants. In these settings, the benefits of long-term PPI will outweigh any increase in relative risk of gastric and oesophageal adenocarcinoma. Nevertheless, this study may still have clinical implications for many individuals. Long-term PPI therapy is exceedingly common and has further increased in recent years, and is often used for off-label indications . The finIn conclusion, this large population-based cohort study indicates that discontinuation of long-term PPI therapy is associated with a decreased risk of both gastric adenocarcinoma and oesophageal adenocarcinoma, although residual confounding cannot be excluded. In the absence of a clear indication for long-term PPI therapy, the need for PPI therapy should be reconsidered to avoid unnecessary cases of gastric and oesophageal adenocarcinoma.Supplementary file 1 (PDF 232 kb)Below is the link to the electronic supplementary material.


To the Editor:For decades, the interest towards glia cells and their contribution to central nervous system (dys)function has been rising. This conceptual expansion opens new avenues to disentangle pathophysiological mechanisms underlying psychiatric illnesses. Amongst the glia cells, oligodendrocyte progenitor cells (OPCs)—also identified as NG2-glia—play a fundamental role in brain function and plasticity, via interaction with glial and neuronal cells, and modulation of neurotransmission . AlthougIn a study published in this journal, Tanti et al. 2021  analysedWhilst providing evidence that CA leads to long-term changes in PNNs, at least in those individuals that have MD, the authors did not clarify how OPCs are implicated in this phenomenon. First, using a single-nucleus transcriptomics approach, the same group previously reported a significant dysregulation in gene expression in BA9 of major depressive (MD) patients compared to CON, independently of early life experiences. Many cell types were affected in MD, but the most prominent dysregulation was observed in the OPC transcriptome. However, in the present study only MD-CA displayed changes in PNN-mRNA in OPCs. If one now tries to reconcile these findings, the question of whether changes in OPC transcriptional profiles are dependent on region and previous aversive experiences will inevitably arise. Or does this further disclose the intrinsic heterogeneity of the OPC population?  In otherPDGFRa, without counterstaining of the soma/processes, thus making it difficult to determine if, for instance, PV-OPC proximity entailed contact or might be purely related to paracrine functions exerted by NG2-glia on PNNs and PV interneurons  cells in BA11, BA12 and BA32 of depressed suicide subjects with a history of CA (DS-CA) compared to CON [+ and APC+) [The role of NG2-glia in stress-related mental disorders is still controversial. Birey and colleagues  reportedd to CON . This rend APC+) , suggest+/CC1+) decreased, compared to CON [As well as post-mortem assessments in humans, preclinical studies provide heterogeneous results on the effect of stress on OPCs. In mPFC of mice exposed to ELA in the form of maternal separation (MS), NG2-glia increased and mature OLs (Olig2d to CON . Prolifed to CON . In contd to CON  found thd to CON . In a did to CON  and decrd to CON  OPCs dend to CON . Reductid to CON , 7. Inted to CON . Taken t