Adequate periprocedural anticoagulation is important to prevent complications like transient ischemic attack, stroke, severe esophageal injury, and pulmonary vein stenosis. The aim of this meta-analysis was to compare uninterrupted anticoagulation therapy with interrupted anticoagulation therapy for patients with arrhythmias undergoing catheter ablation. The current meta-analysis followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Several online databases were searched, such as PubMed, Cochrane Library, and Embase, to search for relevant randomized controlled trials (RCTs). The primary outcome included thromboembolic events. Secondary outcomes included major bleeding events and minor bleeding events. A total of eight RCTs were included in the current meta-analysis, encompassing a total of 3893 patients. No significant differences were reported in relation to thromboembolic events , major bleeding events  and minor bleeding events  between the two study groups. This meta-analysis did not find any conclusive evidence for the absence of any difference between the two strategies.  Atrial fibrillation is one of the most common arrhythmias in adults, and its prevalence is predicted to be increased significantly during the next two to three decades . Rhythm Previous studies have demonstrated that nearly 5-7% of patients undergoing atrial fibrillation catheter ablation experience periprocedural complications -5. Some During catheter ablation, the continuation of oral anticoagulation therapy presents a decision that needs to consider the procedure and the risk of thromboembolic and bleeding events . Thus, eMethodologyThe current meta-analysis was conducted by following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.Data Sources and Search StrategySeveral online databases were searched such as PubMed, Cochrane Library, and Embase to search for relevant randomized controlled trials (RCTs). Searching was performed from inception to 25th September 2022 without putting restrictions on language and year of publication. The following key terms and Medical Subject Headings (MeSH) terms were selected: “interrupted oral anticoagulant”, “uninterrupted oral anticoagulant”, “randomized control trial”, “atrial fibrillation”, and “catheter ablation”. A reference list of all included RCTs was also inspected.Selection CriteriaIn the current meta-analysis, we included RCTs that compared interrupted anticoagulant and uninterrupted anticoagulant therapy in adult patients with atrial fibrillation undergoing catheter ablation. We included all comparisons of uninterrupted and interrupted oral anticoagulants, including any modalities of interruption . We excluded observational studies, non-randomized studies, case reports, and case series. The primary outcome included thromboembolic events. Thromboembolic events were assessed based on clinical parameters. Secondary outcomes included major bleeding events and minor bleeding events. These outcomes were defined by each RCT.Study Selection and Data Extraction ProcessThe selection of studies was performed by two authors independently. Firstly, the abstract and titles of all publications retrieved in the electronic search were reviewed, followed by the full-text screening of potentially relevant studies. In case of any disagreement between the two authors, the issues were resolved through discussion and the involvement of the third author. Data extraction was done by two authors independently using a predesigned form. Data extracted from the selected studies included the name of the author, year of publication, sample size, characteristics of patients, and outcomes. Data extraction forms of both authors were compared, and any disagreement was resolved by discussion. Data were entered into RevMan Software.Quality AssessmentThe risk of bias assessment of all included studies was performed by two independent authors using the Cochrane risk of bias assessment tool. For each study, six domains were assessed including selection bias, detection bias, performance bias, reporting bias, attrition bias, and other potential sources of bias.Data Synthesis and AnalysisThe analysis was performed using Review Manager (RevMan) version 5.4.0 . Risk ratio (RR) was computed for each outcome along with a 95% confidence interval (CI) using Mantel-Hanszel random effect model and presented using forest plots. A RR>1 shows an elevated risk of outcome in an interrupted oral anticoagulant group, whereas a RR<1 shows a higher risk in the uninterrupted oral anticoagulant group. A p-value of less than 0.05 was considered significant. Statistical heterogeneity was assessed using the I-square statistics, and a p-value less than 0.1 showed significant heterogeneity.ResultsFigure Risk of Bias AssessmentFigure Risk of Thromboembolic EventsFive studies reported thromboembolic events between interrupted and uninterrupted anticoagulant therapy in patients with atrial fibrillation undergoing catheter ablation -18. The Risk of Major Bleeding EventsAll included studies compared major bleeding events between interrupted and uninterrupted anticoagulant therapy in patients with atrial fibrillation undergoing catheter ablation -20. No sRisk of Minor Bleeding EventsFive studies assessed minor bleeding events between interrupted and uninterrupted anticoagulant therapy. No significant difference was reported between the two groups in relation to minor bleeding events , as shown in Figure Subgroup AnalysisTable Of the eight studies that compared major bleeding events between interrupted and uninterrupted anticoagulant therapy, one study used VKA in both arms; six studies utilized DOAC in both arms, and one RCT used VKA in one arm and DOAC in the other arm. We compared these three subgroups, and subgroup analysis showed significant differences between the sub-groups .DiscussionThis meta-analysis was conducted to compare uninterrupted anticoagulation therapy with interrupted anticoagulation therapy for patients with arrhythmias undergoing catheter ablation. No significant difference was found between uninterrupted and interrupted anticoagulation therapy in terms of thromboembolic events, major bleeding events, and minor bleeding events. Ottoffy et al. discussed pooled analysis from RCTs, and no difference was found between uninterrupted and interrupted strategies in thromboembolic events . HoweverOne of the major complications of atrial fibrillation is thromboembolic events. The low incidence of thromboembolic events with uninterrupted and interrupted anticoagulant therapy reported in the previous meta-analysis is reassured in the current meta-analysis.Regarding thromboembolic events, Biase et al.  was the Healthcare professionals experience dilemmas when deciding to continue or withhold oral anticoagulants for individuals with atrial fibrillation having catheter ablation procedures . InterruIn individuals with atrial fibrillation undergoing catheter ablation, certain evidence support one strategy over another strategy. We were unable to offer the medical community a strong recommendation due to this ambiguity. However, a detailed examination of the kinds of anticoagulants utilized and how these medications were discontinued is merited. A completely interrupted strategy was linked to a lower likelihood of pre-procedural thromboembolism without enhancing the likelihood of major bleeding events, especially in individuals with long-standing persistent atrial fibrillation, according to the largest study utilizing vitamin K antagonists that contributed the most events to this meta-analysis. For RCTs that used minimal interruption and skipping doses of anticoagulants, the low rate of major bleeding and thromboembolism events can be accredited to the strategic nature.The current meta-analysis has certain limitations. Firstly, the heterogeneity observed in the study outcomes could have limited the findings of this meta-analysis. We did subgroup analysis to explore the heterogeneity where we stratified groups by the type of anticoagulant yielded varied results for study outcomes. However, the findings of this meta-analysis should be interpreted with caution because of the smaller sample size of different meta-analyses and the availability of too few studies in each subgroup arm. Moreover, several studies have less than five events, and those estimates are most unreliable. In the future, more randomized control trials are required with large sample sizes to enhance the power of the findings, which will make them more generalizable.In conclusion, the meta-analysis found that the risk of thromboembolic, major bleeding, and minor bleeding events was not significantly different between patients who received interrupted anticoagulant therapy and uninterrupted anticoagulant therapy. However, due to the low sample size in the majority of studies included in this meta-analysis, the findings need to be interpreted with caution. In the future, multi-centered RCTs need to be conducted that include a larger sample size that can provide more precise findings helping healthcare professionals to select the best treatment option.


Cachexia is an inflammatory and metabolic syndrome of unintentional weight loss through depletion of muscle and adipose tissue. There is limited knowledge of how chronic use of non-steroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids affect cachexia development. The purpose of this study was to investigate associations between prior long-term use of NSAIDs or glucocorticoids with cachexia incidence and post-diagnosis weight loss progression in a retrospective cancer patient cohort.Of 3,802 lung or gastrointestinal cancer patient records, 3,180 comprised our final cohort. Patient demographic information, tumor qualities, medication histories, and comorbidities were assessed. Cachexia was defined as having developed prior to oncologic treatment. Statistical evaluations included categorical, multivariate logistic regression, and log-rank survival analyses. Development of substantial post-diagnosis weight loss was calculated and interpreted for patients without cachexia at diagnosis.P<0.0001). In multivariate analyses, NSAID medications demonstrated a 23 percent reduction in cachexia incidence likelihood . Patients without cachexia at diagnosis were significantly more likely to develop substantial post-diagnosis weight loss from pre-diagnosis use groups of glucocorticoids  or NSAIDs .Chronic prior use of any NSAID or glucocorticoid medication was associated with approximate absolute and relative reductions in cachexia incidence at diagnosis of 10 and 25 percent (Our findings suggest a protective effect of prior anti-inflammatory medications, primarily NSAIDs, against manifestations of the cachexia phenotype at cancer diagnosis. These observations support further exploration of potential therapeutic benefits from anti-inflammatory medications early in cancer management. Cancer-associated wasting, known as cancer cachexia, poses a significant challenge in the management of patients across many primary malignancies. While weight loss is the hallmark of this syndrome, cachexia is further characterized by maladaptive inflammatory signaling through modulation of pathways including JAK/STAT (often through IL-6 cytokines) and both NF-kB and MAP kinase (through TNF-α induction) –4. TheseMultiple pre-clinical and clinical trials have suggested potential benefits in application of non-steroidal anti-inflammatory drugs (NSAIDs) and glucocorticoid medications in the treatment of cachexia-associated weight loss , 8–11. TThe aforementioned trials have been insufficient to validate the inclusion of NSAIDs or glucocorticoids in cachexia treatment guidelines , 13, 14.2, unintentional weight loss greater than 5% of total weight within 6 months prior to cancer diagnosis led to a classification of demonstrating cachectic pre-treatment weight loss. In patients with a BMI < 20 kg/m2, unintentional weight loss greater than 2% within 6 months prior to cancer diagnosis led to the same classification. Patients with stable weight, purposeful weight loss, or weight gain were not categorized as having cachectic weight loss.Cachexia was retrospectively evaluated for the total cohort through review of patient medical records in which weight measurements were routinely documented at each office visit. The assessment of cachexia for each patient was supplemented by vital signs, physician notes, and dietitian notes specifically at the time of cancer diagnosis but before any treatment had been administered. The international consensus definition of cancer cachexia served as the basis for our classification of cachexia at diagnosis . In patiTo define significant weight loss in patients who did not present with cachexia at diagnosis, we applied criterion of reaching 5% weight loss within any 6-month period that ended within 6 months post-diagnosis.Medication prescription and administration logs were obtained through extraction of health system medical records. Patients were placed into prior medication use groups if they were prescribed or administered the selected medications for chronic use prior to cancer diagnosis. We defined chronic use medications as daily medications started at least 3 months before the cancer diagnosis date. Stringent criteria were applied in the selection of medication dosage and formulation type, and any acute or subacute intervention was excluded. Median time from initial medication use per patient to their cancer diagnosis was calculated.Chi square testing determined categorical associations between prior medication use groups and cachexia at diagnosis, as both were recorded as binary variables for each patient. The primary control group consisted of patients without chronic use of anti-inflammatory medications  prior to their cancer diagnosis. An additional control group, patients positive for chronic metformin use and negative for chronic anti-inflammatory medication use, was included in categorical testing. Metformin was selected due to its classification as an unrelated medication class and prevalent chronic use.Multivariate logistic regressions were conducted to consider potentially interfering patient and tumor factors in our evaluation of relationships between anti-inflammatory medication use and cachexia incidence at diagnosis. Cachexia incidence at diagnosis functioned as the binary dependent variable. Patient factors included as covariates were age at diagnosis, sex, race, alcohol history, tobacco history, and Charlson Comorbidity Index. Tumor factors included were primary site and stage. Prior chronic medication use was included as a binary covariate, with null use denoting patients without prior use of any anti-inflammatory medications. Investigated. Overall survival differences between each medication use group versus the medication null group were estimated using the Kaplan-Meier method and evaluated statistically using log-rank testing for the total, cachectic, and noncachectic cohorts.Associations between prior medication use and significant post-diagnosis weight loss in the non-cachectic cohort were evaluated through chi square testing and multivariate analyses. Multivariate analyses included all covariates from the previous multivariate analyses , with the addition of systemic, surgical, radiation interventions as binary covariates. To visually represent weight loss progression in patients non-cachectic at diagnosis, Kaplan-Meier curves were created with significant post-diagnosis weight loss within as the event investigated and days from diagnosis to this event as the time variable.All statistics were conducted with the alpha level of 0.05 defining statistical significance on SPSS Statistics for Mac Version 28.0.1.1 .After applying exclusion criteria, the database was composed of 3,180 consecutive cancer patients seen at the UT Southwestern Medical Center diagnosed between 1/1/2006 and 12/31/2013. Patient and tumor characteristics for the total cohort, as well as sub-cohorts defined by cachexia status at diagnosis, are presented in P=0.0052). Prior chronic use of any NSAID had 10.25% absolute and 28.36% relative decreases in cachexia incidence compared to patients who did not take anti-inflammatory medications prior to diagnosis (P<0.0001). Patients with prior chronic metformin use did not have significant reductions in cachexia incidence . Use of any NSAID prior to diagnosis was associated with decreased likelihood of cachexia incidence by 23.0%  and did not reach a significant difference within the cachexia cohort . Patients with prior NSAID use had median survival times significantly lower than their medication-null counterparts in the non-cachexia group  and did not reach a significant difference for the cohort of patients with cachexia at diagnosis . Similar findings were observed across specific NSAID groups and are further demonstrated in Prior to the review of the effect of anti-inflammatory medications on non-cachectic- and cachectic-specific survival, median survival time was determined to be 23 months  in the total patient cohort, 14 months  in patients with cachexia at diagnosis, and 31 months  for patients without cachexia at diagnosis. Patients with prior use of glucocorticoid medications had median survival times significantly lower than their medication-null counterparts in the non-cachexia group  and 51.75% (P=0.0059) of non-cachectic patients with prior NSAID or glucocorticoid use, respectively, that met the post-diagnosis weight loss cutoff. Similar findings were observed across specific NSAID groups  increased likelihood of meeting criterion for significant post-diagnosis weight loss by 6 months. Prior use of any NSAID medication similarly predicted for 41.1%  increased likelihood of reaching the same criterion (Within the same cohort of 2003 patients who did not meet cachexia criteria at diagnosis, multivariate analyses considering patient, tumor, and treatment factors, prior use of any glucocorticoid medication associated with a 45.2% (Odds ratio=1.452; 95% CI=1.065, 1.979; In this study, we investigated relationships between prior chronic use of NSAID and glucocorticoid medications with cachexia at cancer diagnosis. Multivariate analyses demonstrated a particularly relevant 25% reduction in likelihood of cachexia presentation associated with the NSAID medication class that was independent of potentially confounding patient and tumor characteristics, including comorbidities. Furthermore, our survival and post-diagnosis cachexia analyses are consistent with a delay in cachexia progression attributable to these medications. Although retrospective studies cannot establish causality and are vulnerable to sampling bias, we applied statistical rigor to our dataset through the application of multiple analytical methods and subdivided our cohort to decrease bias and reduce error in our investigation. The novel focus of this study on use of NSAIDs and glucocorticoids prior to cancer treatment intervention primarily highlights NSAIDs for further investigation of therapeutic potential early in cachexia-specific cancer management.Our decision to further investigate the therapeutic potential of NSAID and glucocorticoid medications stemmed from the mechanistic link between these medications and cachexia, as well as further evidence from animal models  and clinThe central mechanism of NSAIDs is inhibition of COX-1 and COX-2 enzymes, which are expressed in almost every human tissue . AlthougPivotal to progression of cachexia is the combination of hypophagia and excessive energy expenditure that promote a debilitating state of catabolism. Although multifactorial in nature, hypophagia observed in cancer cachexia is understood to be primarily generated through the dysregulation of hypothalamic-pituitary axis function by characteristic inflammatory cytokines including TNF-α, IL-1β, and IL-6 , 30. ProIn addition to negative overall metabolic flux, functional decline in cachexia is largely attributable to skeletal muscle wasting from systemic catabolic and inflammatory signaling . In multCancer cachexia remains a critically undertreated component of disease burden across almost every cancer type. Factors contributing to this deficiency include limited options available for management and identification. Although cancer patients are well known to undergo weight loss from treatment-induced anorexia, cachexia uniquely intensifies energy expenditure through malignant metabolic and inflammatory mechanisms.The evaluation of relationships between retrospective medication was carefully considered, as chronic use of any anti-inflammatory medication implies a comorbid condition. This implication prompted our application of multivariate analyses to simultaneously control for comorbidity, as represented by Charlson Comorbidity Index in our cohort, in addition to a multitude of patient and tumor factors. The significant 23% decreased likelihood of cachexia incidence attributable to prior NSAID use interpreted through this method suggests these medications might currently be underutilized in cachexia management and prevention.On initial evaluation, our results demonstrated a survival detriment observed across medication use groups primarily in patients without cachexia at diagnosis. Further evaluation of non-cachectic patients revealed an approximate increase of 10% in the incidence of significant weight loss within 6 months post-diagnosis in patients with prior use of NSAIDs or glucocorticoids. In this cohort of patients without cachexia at diagnosis, multivariate analyses considering interfering patient, tumor, and treatment factors found significant increases in likelihood of post-diagnosis weight loss of more than 40% in NSAID and glucocorticoid use groups. If these medications indeed suppressed the inflammatory ramifications of cachexia-inducing tumor phenotypes prior to diagnosis, it is possible that the reduction of non-cachectic survival represents a progression of cancers which would have classified patients as having cachexia at diagnosis in the absence of prior NSAID or glucocorticoid use. Ultimately, the observed reduction in overall survival within non-cachectic patients with prior NSAID or glucocorticoid use in combination with their increased burden of post-diagnosis weight change potentially represents a delay in cachexia progression by these medications.The findings of this study substantiate further investigation into cachexia-specific intervention with these medications, primarily NSAIDs, early in cancer development. The clinical outcomes we observed are consistent with the biomolecular reasoning that cachexia progression would possess vulnerability to this medication type, which specifically mitigates inflammatory mechanisms of the syndrome not attributable to the body tissue wasting observed consequently to oncologic therapies. Individuals with elevated cancer risk or unexplained weight loss may benefit from these medications prophylactically, as they could potentially blunt cachexia development before patients are burdened by sequelae from cancer progression and treatment. Deciphering the clinical merit contributed by medications with anti-inflammatory properties in this context can alter their prioritization in treatment protocols and physician judgement for managing patients with chronic conditions simultaneously at risk for developing cachexia-inducing tumors.The patient data supporting the conclusions of this article is deidentified through statistical interpretation. The raw data can be made available, without undue reservation, upon reasonable request to the corresponding authors.SO, PI, and RI designed and conceptualized study. SO, BG, and AG participated in data curation. SO conducted formal analyses. PI RI contributed to funding, project administration, and supervision. SO wrote the original draft. All authors participated in review and editing of this project for final publication. All authors contributed to the article and approved the submitted version.This work was supported by National Institutes of Health [P30 CA142543]; Burroughs Wellcome Fund Career Awards for Medical Scientists [1019692]; American Cancer Society grant [133889-RSG-19-195-01-TBE]; Cancer Prevention and Research Institute of Texas [RP200170]; V Foundation Scholar Award [V2019-014]; and American Gastroenterological Association Scholar Award [2019AGARSA3].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.


Primary nasopharyngeal tuberculosis (NPTB) is a rare disease but should not be missed as one of the differential diagnoses for cervical lymphadenopathy or nasopharyngeal mass. We describe a case of a 38 year old lady, who presented with bilateral cervical lymphadenopathy associated with intermittent fever. Nasoendoscopy examination and computed tomography scan of the neck revealed a centrally located mass predominantly at the left posterior nasopharyngeal wall without obliteration of both fossae of Rosenmuller. Typical histopathological features of necrotizing granulomatous lymphadenitis together with the common clinical presentation of cervical lymphadenopathy and nasoendoscopy findings of nasopharyngeal mass conclude the diagnosis of nasopharyngeal tuberculosis. With anti-tuberculous therapy, the cervical lymphadenopathy and nasopharyngeal mass were completely resolved. Nasopharyngeal tuberculosis is an uncommon disease with great diagnostic challenges and with early diagnosis and adequate treatment, NPTB carries a good prognosis with complete disease resolution. Tuberculosis (TB) is known to be one of the oldest infections in the history of humankind, which has caused significant morbidity and mortality since it was first recorded. According to the World Health Organization (WHO) Tuberculosis Report 2020, Mycobacterium tuberculosis had blighted about 10 million people worldwide in 2019  and Eustachian tube . Blood parameters were within normal limits with the erythrocyte sedimentation rate (ESR) value of 14 mm/hour and a negative sputum acid-fast bacilli (AFB) staining. Chest radiography showed normal lung findings. Fine needle aspiration (FNAC) was performed over the left posterior triangle lymph node and showed features consistent with the necrotic lymph node. However, tuberculous lymphadenitis or tumour necrosis cannot be excluded from the cytological findings. Therefore, a contrasted enhanced computed tomography (CECT) scan was done and revealed a well-defined enhancing hypodense lesion, predominantly at the left posterior nasopharyngeal wall, measuring 8.2mm x 6.2mm, without any extension towards bilateral FOR , 2b. There were also enlarged rim-enhancing hypodense lesions over bilateral level 2 and left level 5a of the neck . An excision biopsy of the left posterior triangle lymph node and a biopsy of the nasopharyngeal mass was performed under general anaesthesia. Histopathological examination (HPE) revealed epithelioid cell granulomas with central areas of necrosis surrounded by epithelioid cells mixed with lymphoplasmacytic cell infiltrates and occasional Langhans type multinucleated giant cells . No fungal elements, atypical or malignancy cells present. Surprisingly, the Ziehl-Neelsen (ZN) stain for acid-fast bacilli was negative. A further study performed on the paraffin-embedded tissue for mycobacterium (MTB) polymerase chain reaction (PCR) was also negative for acid-fast bacilli. Given the histopathological findings showing typical features of necrotizing granulomatous lymphadenitis together with the clinical presentation, a diagnosis of nasopharyngeal tuberculosis was made. She was referred to the chest clinic at Hospital Sultan Abdul Halim, Sungai Petani, for the commencement of directly observed treatment short-course (DOTS) for nasopharyngeal tuberculosis.She was treated with an anti-tuberculous therapy regime consisting of Isoniazid, Rifampicin, Ethambutol, Pyrazinamide and Pyridoxine for 2 months during the intensive phase followed by 4 months of Isoniazid and Rifampicin during the maintenance phase. Upon completion of her intensive phase of treatment, her neck swelling had completely resolved, and the nasopharyngeal mass was no longer visualized.Tuberculosis (TB) is a communicable disease that remained one of the leading causes of mortality worldwide. It can manifest particularly in either lung (pulmonary) or other sites (extrapulmonary). About 10-20% of immunocompetent and more than 40% of HIV-positive individuals were affected by extrapulmonary TB. Head and neck TB account for about 10% of extrapulmonary TB and the most commonly involved are the cervical lymph nodes,4. Higheth cranial nerve palsy and sleep apnea. The nasopharyngeal area, being an area of impact by inhaled air is easily overwhelmed by tuberculous droplets which then results in primary nasopharyngeal infection,5. The c. The naseep apnea. In addieep apnea. Large 2Numerous differential diagnoses should be considered when dealing with nasopharyngeal mass. Adenoid tissue, which usually disappears by the age of 16 years old, may be persistent in adults and mimic the appearance of a nasopharyngeal mass. Its per4 bacilli per slide to be present and it has a low sensitivity which ranged only from zero to 44% (Computed tomography (CT) and magnetic resonance imaging (MRI) scan are helpful tools in illustrating the sites, pattern and disease extension, as some radiological characteristics might help in distinguishing between NPTB, malignancies and other differentials. Common radiological findings include polypoidal mass and mucosal thickening in the nasopharynx without extension to the prevertebral muscle, skull base, nasal cavity and oropharyngeal area. Evidence of small caseous necrosis within the nasopharyngeal lesion is one of the valuable hints towards NPTB. Howevero to 44% . PCR analysis is usually reserved in highly suspicious cases of TB but with a negative for Ziehl-Neelsen stain and a negative bacterial culture. In comparison to Ziehl-Neelsen staining, PCR sensitivity was much higher ranging from 75% to more than 95% with a specificity of more than 95% in smear-positive TB. However, its sensitivity reduces to less than 90% in smear-negative TB. As shown in our case, possibly low sensitivity of Ziehl-Neelsen staining led to negative staining and negative bacterial culture for TB as a formalin-stained tissue sample was used for culture. Thus, typical histopathological findings confirmed the diagnosis even though not supported by other routine investigations.Anti-tuberculous regime with 2 months’ intensive phase of Isoniazid, Rifampicin, Ethambutol and Pyrazinamide and 4 months maintenance phase of Isoniazid and Rifampicin were shown effective against both NPTB and pulmonary TB(NPTB carries a good prognosis and complete resolution is expected with adequate and optimal medical treatment. Fortunately, no cases of anti-tuberculous drug resistance or treatment failures were reported. As demonstrated in our case, the complete resolution of symptoms as well as nasoendoscopy findings was achieved after the intensive phase of anti-tuberculous treatment. Nasopharyngeal tuberculosis is an uncommon disease with a great diagnostic challenge as many differential diagnoses need to be considered, particularly NPC, lymphoma and other malignancies. In addition, a wide range of clinical manifestations and less disease awareness might lead to the possible delay in the diagnosis and treatment. The presence of cervical lymphadenopathy with or without other associated symptoms should be followed by thorough ENT assessment, examinations and confirmation by histopathology and bacterial culture if needed. With early diagnosis and adequate treatment, NPTB carries a good prognosis with complete disease resolution.


Adoptive cell therapies expressing TCRs that recognize tumor specific pHLAs take advantage of the unique selectivity and avidity of TCR: pHLA interactions. More recently, additional protein binding domains binding to pHLAs, known as TCR mimics (TCRm), were developed for tumor targeting of high potency therapeutic modalities, including bispecifics, ADCs, CAR T and -NK cells. TCRm compounds take advantage of the exquisite tumor specificity of certain pHLA targets, including cell lineage commitment markers and cancer testis antigens (CTAs). To achieve meaningful anti-tumor responses, it is critical that TCRm compounds integrate both, high target binding affinities and a high degree of target specificity. In this review, we describe the most advanced approaches to achieve both criteria, including affinity- and specificity engineering of TCRs, antibodies and alternative protein scaffolds. We also discuss the status of current TCRm based therapeutics developed in the clinic, key challenges, and emerging trends to improve treatment options for cancer patients treated with TCRm based therapeutics in Oncology. When targeting conventional cell surface antigens with high potency modalities, including chimeric antigen receptor T-cells (CARTcells), bispecifics or antibody drug conjugates (ADCs), frequent onset of on-target, off-tumor toxicities were reported, caused by the expression of the target antigen on normal tissues. These circumstances limited the therapeutic exposure levels of most high potency compounds administered to solid tumor patients and consequently, their anti-tumor activities. The development of dose limiting toxicities on normal tissues significantly reduced the number of high potency compounds advancing to late-stage clinical trials in solid tumors [reviewed in –4]. To oRecently, this therapeutic hypothesis was clinically validated by the unprecedented clinical response rates of uveal melanoma cancer patients treated with a CD3 bispecific targeting the pHLA target gp100, known as T cell receptor mimic (TCRm)-CD3 bispecific compounds. Gp100 represents a lineage commitment marker gene upregulated in melanoma cells of cancer patients, During a productive immune response of the adaptive immune system, two different pathways are concomitantly engaged to induce immunity towards foreign pathogens or self-antigens. These pathways are known as cellular immune response, carried out by T cells and their T cell receptors (TCRs) and the humoral immune response, which is controlled by B cells and mediated by antibodies (Abs) produced by them. In general, cell surface antigens are recognized by Abs, whereas most intracellular targets are recognized by TCRs when presented as pHLA complexes. Two classes of T cells harboring distinct effector mechanisms can be differentiated based on the expression of either CD4 (helper T cells) or CD8 (killer T cells) co-receptors. CD4+ T cells recognize antigens in the context of MHC II and orchestrate the adaptive immune response by secretion of cytokines with pro-inflammatory, chemotactic or immunosuppressive properties. CD8+ T cells recognize antigens bound by MHC I complexes and carry out direct cytotoxic killing of tumor cells [reviewed in ]. The TC3 - 106-fold difference is that TCRs engage with their targets in higher order TCR-clustering, forming a highly controlled, multivalent receptor- target binding complex, with several hundred copies of a TCR bound to an equal number of their respective pHLA targets. Such TCR-pHLA interactions occur in spatially confined, 3 dimensional structures at the interface between T- and tumor cells, known as the immunological synapse. The formation of such multimeric target receptor complexes greatly increases the overall binding avidity and after reaching certain threshold levels, trigger TCR signaling and ultimately tumor cell killing.One key feature distinguishing the interactions between antibodies and TCRs with their respective target antigens are their relative low target binding affinities. TCRs bind to their pHLA targets with apparent binding affinities ranging from 1 to 100 uM , 14, whivia somatic hypermutation, class switching and a clonal selection process resulting in continuously increasing antibody affinities. The impact of increasing the affinities and overall avidities of antibodies on their pharmacological properties have been studied intensively and were summarized recently  induction and inflammatory reactions in clinical settings [reviewed in ]. To achst order avidity) and trigger a divers set of biological anti-tumor responses via their unique Fc gamma receptor  engagement. The responses include the activation of immune effector cells such as macrophages , dendritic- and NK cells  among other mechanisms , in parvia the CAR T approach, a non-immune Fab library was used to isolate a TCR-like Fabs against HLA A1 (MAGE-A1). Subsequent rounds of affinity maturation using a combination of light chain shuffling and heavy chain mutagenesis was employed to generate an anti-HLA-A1 (MAGE-A1) TCRm Fab, which was subsequently cloned into a CAR backbone. High- and low affinity Fabs binding to MAGE-A1 were fused to the FcεRIg chain signaling molecule and construct were transduced to T cells (For targeting of pHLAs  T cells . In addi T cells . These evia a (Gly4Ser)3 spacer domain in conjunction with an immunoglobulin k-leader sequence. The CAR consisted of a CD28 transmembrane and cytoplasmic signaling domains along with the CD3ζ signaling domain , which is present in about 35% of patients with acute myeloid lymphoma (AML). The yeast display library was generated using the full repertoire variable region gene fragments isolated from naïve, human splenic B cells. Yeast display allows for the selection of high affinity binders by increasing the pHLA binding stringency based on scFv binding to the target pHLA complex on the yeast cell surface by flow cytometry. The diversity of the scFv libraries screened can reach 1 x 10t clones . The scFt clones . Since bl models . Combine3, are below the threshold number required for conventional cell surface antigens to internalize sufficient amounts of cytotoxic payloads to induce tumor cell death  and maytansine payloads, both of which rely on medium to high expression levels of cell surface antigens in order to induce meaningful anti-tumor responses. Tubulin inhibitors provide an additional level of tumor specificity, as their mechanism of action is cell cycle dependent. Since most normal tissue cells do not undergo active cell division at the rate tumor cells do, low levels of normal tissue expression of the target antigen is tolerated. Conjugates with the more potent, non-cell cycle dependent, DNA targeting class of payloads, including DNA alkylators and crosslinking compounds such as pyrrolobenzodiazepines (PBDs), cyclopropapyrroloindoles (CPIs), duocarmicins, calicheamicin and others were shown to induce regression of tumors expressing the target antigen at low copy numbers [reviewed in ]. HowevePseudomonas exotoxin A  which are generated by V(D)J recombination, forming a total of six flexible loops, known as complementary determining regions (CDRs) that contact HLA class I and the presented peptide  are then cloned to the variable domain of the light and heavy chain of an IgG1 mAb binding to CD3. This diabody based, bispecifc format is grafted onto an effector function-silenced IgG1 Fc domain, that contains additional knob and whole mutation to stabilize the molecule. TCER compounds display picomolar cellular potencies and greatly extended half lives compare to the non-half live extended ImmTac compounds (A similar TCR affinity maturation approach was applied to generate bispecific T Cell Engaging Receptor (TCER) compounds as CD3 bispecifics. For this platform, an ompounds . The molAs shown in When scaling production for manufacturing, antibodies can display variabilities in their expression yields, in their tendencies to form aggregates and in their reliance on disulfide bonds for stability and to maintain appropriate glycosylation, and in most cases, require a mammalian cell expression system. These circumstances triggered a search for alternative protein scaffolds to address these manufacturing liabilities while achieving the same target binding affinity and specificity as antibodies. Previous attempts to generate non-antibody-based binders relied on loop or surface randomization of different protein scaffolds, which are fixed in dimension , 97. SomOne of the key challenges when targeting pHLA complexes is the large number of potential targets that can be recognized by TCR based therapeutics. Therefore, comprehensive profiling of TCR and TCRm compounds is required to achieve high levels of target saturation and specificity. This has become even more relevant after recent reports suggested that the intended target peptides of TCRs or TCRm compounds often differ in only one or two amino acids from unintended, off-target pHLAs that can be present on healthy tissues [reviewed in ].More recently, several high throughput peptide panning methods were described to more comprehensively profile TCR or TCRm compounds for target selectivity during the engineering process. These technologies carry the potential of eliminating off-target binding introduced during the affinity maturation process [In addition, pHLA targets represent a challenge for protein engineering, as only a small area of the binding interface between the pHLA complex is accessible to the binders. In the case of antibodies binding to the WT-1 pHLA complex for example, only between 12% and 27% of the pHLA surface area is directly involved in binding to the IgG binder . An optiThe mechanisms underlying the development of resistance towards treatment with pHLA targeting compounds was studies in various translational studies in the clinic. Several studies conducted with TCR-T cells targeting pHLA targets suggested that pHLA downregulation, as a consequence of mutations acquired during treatment, either reduced pHLA presentation or decreased IFN-y production in tumor cells , 107. SuSome early reports studied the genetic changes associated with the development of resistance towards treatment with TCR-T cells, when administered to melanoma cancer patients with multiple metastatic lesions . The dat+ T cells in bladder tumors treated with anti-PD1 immunotherapy, which exerted killing of autologous tumors ex vivo (To overcome the development of resistance towards TCR based therapies, several approaches are currently being pursued, as outlined below. A common strategy to address pHLA target downregulation is concomitant targeting of multiple pHLA targets that are co-expressed on the same tumor. In this context, multi target binder formats that can accommodate multiple pHLA targets are worth further exploration . Alterna ex vivo . Finally ex vivo . Both en ex vivo  when tesIn conclusion, the lessons from these preclinical and clinical studies investigating the development of resistance towards TCR based therapies have great potential to guide the future clinical and translational development of TCRm based therapies targeting solid tumorsAll authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.We would like to thank Leah V. Sibener and Alejandro Ramirez for critical reading of the manuscript and their helpful comments and discussions.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.


The goal of this study was to investigate the extent of the alveolar-capillary membrane porosity in patients with severe re-expansion pulmonary oedema. The biochemistry of airway fluid of two patients who died of re-expansion oedema was compared to their blood biochemistry. The airway fluid was comparable to plasma, while no blood cells were observed across the alveolar-capillary membrane. The membrane was linked to a fishnet that traps cells on one side, while plasma sieved through. Re-expansion pulmonary oedema is a rare form of acute lung injury, occurring as a complication of sudden lung re-expansion after pleural drainage [1]. Re-expansion pulmonary oedema is a rare form of acute lung injury, occurring as a complication of sudden lung re-expansion after pleural drainage . This poA 44-year-old patient presented with a loculated right empyema thoracis from transdiaphragmatic rupture of a right subphrenic abscess. After failed thoracentesis coupled with a demonstrable visceral pleural cortex on chest computed tomography scan, a single-stage pleural drainage and lung decortication were done via thoracotomy. About 3000 ml of thick pus was drained from both the chest and abscess, and the lung re-expanded fully after decortication. Large amounts of frothy serosanguineous fluid were noted in the patient’s endotracheal tube. Bronchoscopy revealed large amounts of the fluid pouring from left and right bronchial trees. Despite being on a ventilator, 400 ml of airway fluid was suctioned in about 15 min and even more thereafter. Ineffective ventilation due to build-up of fluid in the endotracheal tube resulted in hypoxic cardiac arrest and death (Fig. A 31-year-old male patient presented with a massive right pleural effusion. A chest drain was inserted, draining 800 ml of straw-coloured fluid. The drain was clamped with the aim of draining smaller amounts in a controlled fashion. Unfortunately, the clamp slipped off resulting in sudden drainage of 1500 ml, causing pulmonary oedema that required ventilation. The endotracheal tube rapidly filled up with large amounts of serosanguineous fluid, again resulting in death. The airway fluid and blood biochemical properties were compared (Table First described in 1853 by Pinault . ReperfuDirect comparison between airway fluid and blood biochemical components in our cases demonstrated a massive protein leak through the capillary alveolar barrier. Total protein, albumin and lactate dehydrogenase levels were comparable to serum Table . Both smet al. [Hydrostatic mechanism was previously demonstrated in 5 out of 7 patients by Sue et al. . Using tAs shown in our study, ineffective ventilation due to fluid accumulation in the airway is fatal and extracorporeal membrane oxygenation should be considered.Our study demonstrated an alveolar-capillary membrane that is permeable to plasma, but not to blood cells, in some patients with severe re-expansion pulmonary oedema. Although larger studies have been previously reported, this study adds some understanding to the potentially complex pathophysiological mechanisms of this condition.Written informed consent was obtained from both patients relatives for publication.Conflict of interest: none declared. The data underlying this report will be shared upon request to the corresponding author.Interactive CardioVascular and Thoracic Surgery thanks Paola Ciriaco, Francoise Le Pimpec-Barthes and the other anonymous reviewers for their contribution to the peer review process of this article.


Cardiovascular disease (CVD) has been a prominent global health challenge in the last decade, and many risk factors and outcomes of CVD have been studied in that timeframe. Recent research has explored the association between sugar-sweetened beverage (SSB) consumption and CVD; however, there is a lack of updated reviews regarding SSB consumption impacts on CVD outcomes and the possible mechanisms affecting the disease state. In turn, this review aims to summarize the relevant published research from the last decade regarding linkages between SSB consumption and CVD outcomes and the potential underlying mechanisms, as well as to highlight opportunities for future exploration with respect to those outcomes and mechanisms. In this review, we searched PubMed, Embase, and Web of Science for peer-reviewed articles published from January 2012 to March 2022 regarding SSB consumption and its association with CVD.The results of our search reveal strong evidence that the consumption of SSB is positively associated with increased risks of CVD and that the magnitude of that risk is increased in a dose-dependent manner. These increased risks range from elevated triglyceride levels to inclined risk of CVD-related mortality. Although the depth of the mechanisms responsible for these increased risks have been less explored thus far, there is some evidence supporting SSB implications in cardiovascular factors, including vascular function, coronary artery calcification, triglyceride levels, inflammatory processes, arterial stiffness, and genetic polymorphisms. Sugar-sweetened beverages (SSB) comprise a vast array of liquid drinks, including fruit juices, carbonated sodas, vitamin water drinks, energy drinks with added sucrose, fruit juice extracts, and, most commonly, high-fructose corn syrup . Most ofCVD has been a prominent global health challenge in the last decade . ResearcThe goals of this review were to summarize relevant published research regarding linkages between SSB consumption and CVD and to highlight opportunities for future research to clarify mechanisms behind the CVD outcomes associated with SSB consumption. For this purpose, use is made of risk comparisons of morbidity and mortality outcomes for persons consuming various amounts of SSBs as reported in the literature. These assessments often utilize relative risk parameters and hazard ratios to quantify the findings.Search strategyThree databases were searched for peer-reviewed articles written in English: PubMed, Embase, and Web of Science. The phrase “sweetened beverage*” was mandatory in the title and “sugar” was mandatory in the title or abstract of the searches. Additionally, one or more of the following words was mandatory in the title: “cardi*”, “coronar*”, “heart”, “vasc*”, “myo*”, “infarct*”, “ischem*”, “stroke”, “hypertension”. The purpose of the asterisks (*) was to include any titles with variations of those words. Studies published from January 2012 to March 2022 were included. After accounting for duplicate articles across the three databases, 71 initial articles remained for review. After exclusion of two animal studies, 19 review and systematic review articles, and 26 research articles with non-CVD focuses , 24 meta-analyses and primary articles remained for review. Additionally, six more relevant articles found within the initial 24 studies were included for a total of 30 articles assessed in this review.The impacts of global SSB consumption on CVD occurrence and mortalityThe link between SSB consumption and CVD occurrence and mortality has been studied from several different perspectives. One way was to compare the health outcomes of sugary beverage consumers versus non-consumers . In thisSpecific research on cardiovascular mortalities associated with SSB consumption also supports the SSB-CVD linkage. For example, an analysis of over 100,000 men and women found a 31% higher risk of death from CVD in persons who consumed two or more SSB servings per day versus those who consumed less than one serving per month (p<0.0001) . AnotherAssociations of SSB consumption with specific cardiovascular eventsBecause CVD encompasses a large span of illnesses affecting the heart and vasculature, it is of interest and potential utility to focus more specifically on these varied aspects individually.SSB Impacts on Coronary Vascular DiseaseCoronary-specific diseases have been investigated over the last ten years. The presence or history of myocardial infarction (MI) is one outcome measure by which studies gauge coronary artery disease (CAD). Some evidence suggests an increased risk of CAD for high versus low SSB consumers based on MI risk. One study found in a 22-year span of initially 18,000 participants that the top-quartile of SSB consumers had a 20% higher risk versus the bottom-quartile of developing CAD after accounting for lifestyle variables, including age, smoking, exercise, body weight changes, and diet considerations  ,27. AnotMore limited evidence points to a link between SSB consumption and coronary disease-related deaths. An analysis of the United States National Health and Nutrition Examination Survey (NHANES) across 15 years that included more than 30,000 individuals reported a significant difference in heart disease deaths between the highest and lowest quintiles of SSB consumers  . The datSSB Impacts on StrokeOne aspect of peripheral vascular disease (PVD) regards the potential impact of SSB consumption on stroke. A study of 106,178 women in the California Teachers Study found a significantly higher hazard ratio for stroke in those who consumed one or more servings of SSBs daily versus those who rarely or never consumed SSB  . This asSSB Impacts on Blood PressureThe effects of SSBs on blood pressure have also been investigated. In one study of over 60,000 university students in Iran, individuals in the highest category of SSB intake were at 2.17 times greater risk of developing hypertension versus a low-consumption group  . SimilarMarkers and mechanisms of SSB-CVD linkagesWhile studies investigating SSB-CVD linkages are relatively numerous, few studies address the potential mechanisms involved. These include possible impacts of SSB on endothelial cell properties or vascular function, triglycerides, C-reactive protein (CRP), and other biomarkers. These aspects are considered in this section.SSB Impacts on Vascular FunctionOnly one small study was found that addressed this issue directly by comparing the impacts of SSB versus water consumption on endothelial cell properties in healthy males . This stFollowing a 10-hour fasting interval, 12 male subjects consumed 600 ml of water over a five-minute interval during one visit and 600 ml of a commercial SSB in another five-minute interval at a separate visit . FifteenSSB Impacts on TriglyceridesAnother possible SSB-CVD linkage is its involvement with triglycerides ,12,17. ASSB Impacts on C-Reactive ProteinSeveral studies have reported positive associations between SSB intake and C-reactive protein (CRP) ,15,18. BSSB Impacts on Coronary Artery CalciumA positive association between SSB intake and the amount of coronary artery calcium (CAC) has also been reported . CAC is SSB Impacts on Arterial Stiffness Determined via Pulse Wave Velocity Measurements Arterial pulse wave velocity (PWV) depends on the stiffness of the arteries in which the pulses are traveling with a higher velocity indicating a greater stiffness . Such meOne study used this method to investigate a possible association between fructose consumption, including SSB, and carotid-femoral PWV measurements in over 1000 participants . The resRole of Genetic Variations in the SSB-CVD LinkStudies have shown an association between single nucleotide polymorphisms (SNPs) on chromosome 9p21 and CVD ,46. In aThis study elucidates the opportunity for expansion of research on the association between SSB intake and genetic involvement. Although the study included an appreciable sample size of 1560 participants with CVD events, the population was specific to Hispanics living in Costa Rica . ReplicaThere is strong evidence that the consumption of SSB is positively associated with increased risks of CVD and that the magnitude of those risks are increased in a dose-dependent fashion with SSB intake. Specifically, support exists for increased risk of stroke, myocardial infarction, and CVD-related mortality associated with the consumption of SSB.Research focuses on both CVD outcomes and the mechanisms responsible for the disease state in relation to SSB intake can be expanded to continue the depth of this knowledge. For example, within the vast spectrum of populations consuming SSBs that are affected by CVD, there may be certain ethnicities, races, communities, etc., with significantly worse CVD outcomes. Further investigation into the associations of SSB consumption with CVD outcomes in different populations and continued expansion of the methods in which these studies are performed can improve the current knowledge. Similarly, the current finite research on mechanisms underlying SSB involvement in CVD presents many avenues for future studies. Compounding research into vascular function, coronary artery calcification, triglyceride effects, inflammatory processes, arterial stiffness, and genetic polymorphisms in SSB-consuming participants could increase our understanding of the drinks’ implications in CVD. Examples may include expanded studies with PWV measurements of different SSB-consuming populations or dose-dependent effects of the sugary drinks on genetic variations with CVD associations.This review brings attention to recently studied associations between SSB intake and CVD outcomes and possible mechanisms leading to those outcomes. It also illuminates opportunities for future research in the field. The information highlighted throughout this article may serve to bring awareness to the SSB-CVD linkages and aid in interventions to improve health outcomes.


Ecological theory predicts close relationships between macroclimate and functional traits. Yet, global climatic gradients correlate only weakly with the trait composition of local plant communities, suggesting that important factors have been ignored. Here, we investigate the consistency of climate-trait relationships for plant communities in European habitats. Assuming that local factors are better accounted for in more narrowly defined habitats, we assigned > 300,000 vegetation plots to hierarchically classified habitats and modelled the effects of climate on the community-weighted means of four key functional traits using generalized additive models. We found that the predictive power of climate increased from broadly to narrowly defined habitats for specific leaf area and root length, but not for plant height and seed mass. Although macroclimate generally predicted the distribution of all traits, its effects varied, with habitat-specificity increasing toward more narrowly defined habitats. We conclude that macroclimate is an important determinant of terrestrial plant communities, but future predictions of climatic effects must consider how habitats are defined. Climatic variables are useful but often weak predictors of plant functional trait variation across ecosystems. Here the authors investigate how assigning plant communities to a habitat hierarchy improves the explanatory power of climate-trait relationships at the continental scale. Ecosystem functioning, such as productivity and nutrient cycling, is strongly determined by the functional composition of the plant community6. Functional traits represent species’ life-history strategies7, and are often summarized with a few main, largely independent, axes of variation, such as the fast-slow continuum8, as reflected in the leaf-economics spectrum9, the species’ reproductive strategy10, the plant size spectrum7, and the continuum of collaboration with mycorrhizal fungi11. A foundational, yet globally weakly supported, assumption in trait-based ecology is that the geographical distribution of dominant functional traits in plant communities is shaped by macro-environmental gradients, independently of taxonomy14. Here, we addressed this assumption by studying the consistency of macroclimate-trait relationships among European plant communities.Predicting the effects of a changing climate on the diversity and functioning of the ecosphere requires an understanding of how climate drives the distribution of plant species and ecosystem properties15). At the global scale, however, CWMs have been weakly predicted by linear gradients of climatic conditions13. Yet, in Australia, which can be considered a relatively homogeneous evolutionary unit, climate predicted 43% of the variation in CWMs16, suggesting that global climate-trait relationships might be blurred by different phylogenetic histories when modelled across biogeographic realms. Furthermore, these relationships could also be significantly affected by habitat-specific factors17. When considering local plant communities, CWMs are likely shaped by soil conditions19, microclimate20, human land use21, disturbance22, biotic interactions23 (e.g. herbivory or competition24) and evolutionary history that constrainsthe available species pool25. All of these factors likely interact with macroclimatic gradients. Hence, they may mask the actual effects of climate on local plant communities.The most popular approach to studying climate-trait relationships involves regression analysis between climatic gradients and the community-weighted mean of plant functional traits (CWMs26). Yet, it is unclear whether the strength and expression of climate-trait relationships depend on the thematic resolution (hereafter “narrowness”) of the habitat definition. Analysing climate-trait relationships at different levels of habitat narrowness could help us understand whether these relationships are consistent across both levels (resolution-invariant) and habitats . This understanding might be important for more accurate predictions of the effects of a changing climate on the distribution and functioning of natural plant communities.The classification of plant communities into hierarchically arranged habitat types (hereafter habitats) offers a promising approach to studying climate-trait relationships because it accounts for interfering local factors that define different habitats. Restricting analyses to habitats with similar floristic composition, shared evolutionary history and comparable environmental conditions could disentangle the effects of interacting factors and result in tighter climate-trait relationships, as has been observed in analyses focused on specific habitats (e.g. forest and tundra habitats27), 19 high-resolution bioclimatic variables from the CHELSA database29 and species-level plant trait information from the TRY database31. Each plot was characterized by the CWM of four key functional traits: plant height, specific leaf area (SLA), seed mass and specific root length (SRL) (Table 7) and in the root economic spectrum (SRL11). Each plot was classified to three hierarchical levels of the EUNIS habitat classification33 which include eight broadly defined level 1 habitats (hereafter broad habitats), 40 more narrowly defined level 2 habitats (intermediate habitats) and 216 of the most narrowly defined level 3 habitats (narrow habitats)33. We applied generalized additive mixed-effects models that accounted for spatial dependence among plots to determine the linear effects of the principal components of the European climatic gradients (climate PCs) on the distribution of the four CWMs. We hypothesized that (i) climate has a general effect on CWMs across habitats  using principal component analysis. These four gradients jointly captured 88.4% of the variation in macroclimatic conditions . Similarly, we found that higher temperatures led to higher community plant height in wetland habitats, except for periodically exposed shores (Q6), where temperature was negatively related to plant height. Our results on the habitat-specificity of climatic effects are in accordance with previous findings on the distinct effects of precipitation on vegetation productivity in grassland versus forest habitats in China36. On the global scale, the predictive power of climate for the distribution of community leaf trait composition increased when 14 rather than four broad habitat types were considered37. This finding provides an explanation for why studies in more narrowly defined habitats revealed more pronounced effects of climate on plant traits (e.g. as shown in tundra habitats26) than studies across habitats13. The two community traits that were less well predicted by habitat alone (namely SLA and SRL) were increasingly better predicted by climate when habitats were more narrowly defined. The underlying reason might be that SLA depends more strongly on soil than on climatic conditions38 and that the relationship between SRL and environmental gradients also strongly depends on the habitat considered39. Taken together, these observations suggest that broad habitats include species with different life-history strategies , while more narrowly defined habitats include species with more similar life-history strategies. Consequently, broad habitats might allow for a wider range of alternative climatic responses, while in narrow habitats, species tend to share similar climatic responses. Thus, the effect of climate on the proportion of species with high or low SLA (or SRL) becomes clearer. The two traits that did not follow this pattern, i.e. community plant height and seed mass, are strongly correlated within the global spectrum of plant form and function40 and most strongly determined by habitat type. Community plant height is related to the occurrence of woody species, which markedly depends on land use and is also more strongly determined by soil moisture and nutrients than by broad climatic conditions41.The habitat classification we used is based on floristic and biogeographic characteristics. As a result, plant communities were analysed within homogeneous groups that reflect common adaptations to local environmental features that are either independent of global climatic gradients43. In forest habitats, the decrease in plant height along the Mediterranean gradient could be attributed to the limiting effects of higher temperatures and lower water availability on the growth of canopy trees. The distribution of forest understory traits, however, is fairly independent of precipitation gradients in Europe20. Community height in coastal habitats could be limited by precipitation because dune soils are highly permeable  and the groundwater is not available to most plants44. For SLA, we observed a decline along the Mediterranean gradient , corresponding to the global13 and intra-specific reduction of SLA under drought45. The same arguments apply to the effects of temperature. Wetland communities, for instance, might be able to maintain a higher SLA, even under higher temperatures, because they are not constrained by water availability. Grassland species might benefit from higher SLA in warmer temperatures if they are able to compensate for natural or human-induced disturbances by regrowing relatively quickly. In man-made habitats, the response of SLA might depend on the relative abundance of different life forms, with therophytes being relatively more abundant at higher temperatures46. Community seed mass showed the greatest consistency in climate-trait relationships across all broad habitats. Yet, the increase in seed mass along the Mediterranean gradient was quite unexpected, as seed mass tends to increase with precipitation and decrease with aridity on the global scale13 or is only weakly related to precipitation in Mongolia, China47 and Australia48. In habitats where water availability depends on groundwater, such as coastal salt marshes, coastal habitats and wetlands, community seed mass was independent of the Mediterranean gradient. An explanation might be that larger seeds, which can buffer the survival of seedlings and saplings49, are most important during hot and dry summers. For community SRL, the observed increase along the Mediterranean and temperature gradients refuted our expectations. In coastal saltmarshes, heathland and forest habitats, however, we also observed decreases in SRL along both gradients, suggesting that the effects of macroclimate may be strongly habitat-specific or masked by local factors, such as local soil conditions19. These local conditions were increasingly accounted for by including more narrowly defined habitats in the models of climate-SRL relationships.Although the investigated climate-trait relationships were mostly consistent across broadly defined habitats, we found several habitat-specific relationships. For instance, the increase in plant height along the Mediterranean and temperature gradients could be explained by the longer growing season and higher light availability, which both increase competition for light in warmer regions50 and leaf traits20). We partially accounted for non-linearity by using log-transformed CWMs. Still, our models identified stronger linear effects of climate on community traits compared to global-scale analyses13. Whether the classification into narrower habitats might have strengthened or alleviated any nonlinear or interaction effects has, to our knowledge, not been tested yet. By focusing exclusively on climate, we did not account for important interactions with edaphic conditions (such as evapotranspiration), which are the best predictor for leaf area and maximum plant height distributions across Australia16. Furthermore, it is conceivable that the effects of climate on community trait composition could change at the end of climatic gradients. This assumption could be tested by comparing trait-environment relationships under benign versus extreme conditions39. In forests, we did not differentiate between the canopy and understory layers, although these layers might show contrasting responses to climate20. Finally, we calculated CWMs based on species-level trait averages rather than in situ measurements, which precluded any analysis of intra-specific trait variation. These shortcomings may have diluted the effects of climate on CWMs, but were unavoidable given the large geographic extent of our study.In order to synthesize the effects of macroclimate across a large-scale dataset, we had to accept some shortcomings in our analysis. We focused only on the linear effects of climate and ignored interactions among the different dimensions of climate, although we can assume that the distribution of the four plant traits is also shaped by nonlinear and interaction effects 53 to maximize compliance with species names from the TRY plant trait database30. Fungi, bryophytes, algae, and lichens were removed from the data, resulting in 13,758 vascular plant species.We harmonized species names from the European Vegetation Archive using the Taxonomic Name Resolution Service 5.0 (tnrs.biendata.org). Subspecies and varieties were merged at the species level. Species names were matched to the taxonomic backbone 3.0 of the sPlot global vegetation database30 version 5, which covered species-level mean values of 33 traits for 50,404 species, derived with Bayesian Hierarchical Probabilistic Matrix Factorization. This algorithm predicts individual-level trait values based on the observed trait records, observed trait-trait correlations, and the taxonomic hierarchy reflecting phylogeny55. The root-mean-square error of the z-transformed predicted versus available trait values ranged from 0.07 (seed mass) to 0.2 (SLA) , updated on October 25th, 2021. We used the first three levels of the classification hierarchy, ranging from the highest level 1  through the intermediate level 2  to the lowest level 3 . This classification system included the following broadly defined habitat types at level 1: coastal salt marshes , coastal habitats , wetlands , grasslands , heathlands , forests , inland sparsely vegetated habitats  and vegetated man-made habitats . All intermediate and narrow habitats are listed in Supplementary Data 33 and56.Each plot was assigned to a habitat type based on the classification expert system EUNIS-ESy (European Nature Information System29 at a pixel resolution of 30 arc sec (~1 km) using the cloud-based Google Earth Engine platform. Bioclimatic values were calculated as the average value from all pixels within 500 m from the plot coordinates. Plots with missing climate data in adjacent pixels were omitted from further analyses. Covariation among the 19 bioclimatic variables was removed by a principal component analysis. The first four principal components (PCs) jointly explained 88.4% of the climatic variation  to account for spatial dependence among plots58. For each of the four traits, we created the following five different models: Model 1 included only the fixed-effects of the four climatic PCs without any interactions. Only in this model, we accounted for the different number of observations per habitat type by including only the same number of randomly selected plots from each of the broad habitats . Model 2 included the fixed-effects of the four climatic PCs plus the random effects of the broad level 1 habitat types. Model 3 included the fixed-effects of the four climatic PCs, the random effects of the broad level 1 habitat types plus random interaction terms between the fixed effects of the climatic PCs and the broad level 1 habitat types. Models 4–5 were similar to model 3 except that the broad level 1 habitat types and their random interaction terms were replaced with the intermediate level 2 or the narrow level 3 habitat types and their respective interactions with the climatic PCs. The implementation of all models is documented on GitHub. Model residuals were visually checked and are shown in Supplementary Figs. Prior to the following analyses, all plot-level CWMs were log transformed to approximate normality. We estimated the effects of the four climatic PCs on the CWMs of the four traits using separate generalized additive mixed-effects models that always included a spline-on-the-sphere smoothing term  and habitat type (random effects). The proportion of explained variation was quantified using the marginal and conditional R²59, whose calculation for random-slope models was based on the variance components of the fixed and random effects and whose implementation we adopted from the R script provided by60. To check whether the obtained results depended on the proportion of species with actual trait measurements, we repeated all analyses with models in which the contribution of each plot was weighted according to the summed cover of species with trait values divided by the total plant cover. The results of these weighted analyses were qualitatively similar to the results of the unweighted analyses and are shown in Figs. Supplementary SFor models 3–5, we quantified the effects of climate on trait CWMs, both across and within habitats, with predicted marginal mean regression slopes. All regression slopes were assigned an approximate confidence interval (slope estimate ± 1.96 * standard error) and confidence intervals that did not include zero were considered significant at approximately R61 using the package mgcv for generalized additive mixed-effects models58, emmeans and ggeffects for marginal mean regression slopes63, dismo for geographical stratification of plots64 and ggplot2 for data visualization65.All analyses were conducted in Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary InformationSupplementary Data 1Supplementary Data 2Reporting Summary


ALK rearranged NSCLC is sensitive to ALK tyrosine kinase inhibitors (TKIs) but prone to drug resistance. Meanwhile, ALK rearranged NSCLC has poor response to single immunotherapy. Here we mainly describe the immune escape mechanisms of ALK mutated NSCLC and the role of related biomarkers. Additionally, we collate and evaluate preclinical and clinical studies of novel immune combination regimens, and describe the prospects and perspectives for the  Nearly 80% of lung cancer is NSCLC, one of the leading causes of death due to cancer today , 2. OncoThe oncogenic role of ALK was first recognized with the discovery of the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) fusion in anaplastic large cell non-Hodgkin lymphoma (ALCL) . Since tThe pathogenesis of lung cancer induced by EML4-ALK has been confirmed by evidence, and the rapid development of lung cancer is mainly caused by overexpression of EML4-ALK in type II alveolar epithelial cells through the Clara cell secretory protein (CCSP) or surfactant protein c (SPC) promoter , 11. In Anaplastic lymphoma kinase (ALK) tyrosine kinase receptor has been identified as a therapeutic target in lung cancer. We analyzed the direction of recent research on ALK related articles to retrieve ALK related articles through PubMed for the last decade Figure 2Numerous preclinical and clinical studies have suggested that wild-type ALK is predominantly expressed within the peripheral and central nervous system, with little expression in the remaining sites . BecauseThe intracellular kinase domain of ALK is a 220 kDa cell surface tyrosine kinase receptor that belongs to the insulin receptor superfamily. In addition, the ALK extracellular domain includes a glycine rich region, a low-density lipoprotein class a domain, and two MAM regions (meprin/A5 protein/ptpmu) , 18 Fi–21.At the primary sequence level, EML proteins are composed of a number of WD (Trp-Asp) repeats N-terminal to them and a help (hydrophobic Echinoderm Microtubule-Associated Protein (EMAP) like protein) motif present at the N-terminus, whereas at the tertiary sequence level, EML proteins consist of a trimerization domain (TD), a basic region, and a Tandem Atypical Propeller EML (TAPE) domain . The rolThere are studies showing the role of ALK in regulating innate immunity during lethal sepsis , 24. Mecin vivo in ALCL patients in the early 2000s by Pulford et al., while antibodies against ALK-WT were also found to be present in 91% of patients  responses to eliminate tumor cells. Relevant studies have demonstrated the existence of a precursor pool of anti ALK CD8+T lymphocytes in cancer patients . Some stALK fusion genes not only cause body CTL response and humoral immunity, but also induce the expression of PD-L1 on tumor cells –31. The High expression of PD-L1 and PD-L2 on the surface of tumor cells triggers immune evasion , 35. StuAlthough many findings have shown that the PD-L1 expression rate of EML4-ALK fusion in NSCLC is higher than that of wild type, the poor efficacy of immunotherapy in NSCLC with EML4-ALK fusion is conflicting, and the reason to explain this phenomenon may be the significant difference in study populations and study methods. For example, PD-L1 expression is more variable in ALK fused NSCLC , 41, whiTumor mutational burden (TMB) was defined as the total number of detected, somatic gene coding errors, base substitution, gene insertion or deletion errors per million bases, excluding germline mutations . TherefoMajor histocompatibility complexes I and II (MHC-I and MHC-II) are key players in the activation and antigen presentation of immune cells of the body. MHC-I is encoded by the HLA-A, - B, and - C genes, and it can be interferon-gamma (IFN-g) and inflammatory pathways such as NF-kB induce expression. Several reasons have been attributed to downregulation of MHC-I expression by cancer cells, including loss of B2M, mutation or deletion of HLA genes and defective trafficking to the cell surface . Loss ofin vitro and in vivo experiments, while they can decrease PD-L1 levels on the surface of tumor cells by 75%. This suggests that ALK mutant NSCLC may facilitate cancer initiation by enabling tumor evasion of the immune system through downregulation of HLA expression (T cell immunotherapy is often hampered by limited presentation of tumor specific antigens due to downregulation of human leukocyte antigens (HLA). A recent study found that ALK inhibitors can increase the dosage of HLA associated proteins on the surface of ALK mutated lung cancer cells in both pression . TherefoPredictive biomarkers of current ICIs treatment efficacy besides PD-L1 expression level, major histocompatibility complexes and TMB, but also TME, microsatellite instability high (MSI-H) and mismatch repair deficiency (dMMR), among others –57.The tumour microenvironment (TME) in ALK rearranged cancers refers to the interplay of CD4 + T cells, CD8 + T cells, tumour associated macrophages (TAMs), mast cells and regulatory T cells (FOXP3 +) . PreclinRecently, studies have also pointed out that mismatch repair (MMR) deficiency and microsatellite instability (MSI) could also predict the therapeutic efficacy of tumor immunotherapy to some extent . Defectiin-situ hybridization (FISH), reverse transcription polymerase chain reaction (RT-PCR), next-generation sequencing (NGS). Fish testing has high sensitivity and specificity to ALK fusion gene detection, and is the gold standard for ALK rearrangement detection (The incidence of ALK rearrangement in lung adenocarcinoma (LADC) is higher than that in lung squamous-cell carcinoma (LSCC) , 66. Theetection . Ventanaetection . Therefoetection , 69. Theetection . With thetection , 72. RT-etection .The fusion of EML4-ALK is a unique oncogenic driver mutation in NSCLC, which occurs in approximately 4-8.1% of NSCLC patients , 75. AltBecause ALK rearrangements are relatively uncommon in patients with NSCLC, clinical data on single agent ICI therapy for ALK positive NSCLC are mainly derived from retrospective studies and case reports Table 1.There are mainly several following prospective randomized clinical trials of single agent ICIs in patients with ALK rearranged NSCLC. In the ATLANTIC study , the effTo further improve the survival of patients with NSCLC, studies of combination regimens targeting IC have been successively conducted. The results of a recent study found that the combination of nivolumab and ipilimumab is an effective treatment for patients with metastatic NSCLC . AlthougThere are existing clinical studies on the combination therapy of ALK TKI with ICI, and preliminary data suggest good efficacy Table 2.Checkmate 370 is a clinical trial for previously untreated advanced ALK rearranged NSCLC patients treated with nivolumab in combination with crizotinib. Of the 13 patients enrolled, 5 (38%) had a partial response, however patient enrollment was stopped due to severe drug toxicity episodes . In addiRecently, Patel et al. analyzed the results of a phase IB study of a combination regimen of pembrolizumab plus crizotinib in patients with previously untreated ALK positive NSCLC. The study terminated the trial early because of dose limiting toxicity occurring in four patients (44%), including hepatotoxicity in three patients . In a reAs immunotherapy advances, more research teams have pursued the use of ICIs in standard chemotherapy regimens, expecting that more cancer patients will benefit from immunotherapy. Combining immunotherapy with chemotherapy may increase immune system activity through the immune effects of chemotherapy , which, But the effect of immunotherapy combined with chemotherapy for ALK rearranged NSCLS remains undefined. Preliminary study data on this treatment option are already available: IMpower150 and IMpower130 Table 3.Anti-angiogenic agents induce tumor immune escape, and as immune checkpoint inhibitors are applied to patients with advanced NSCLC, antiangiogenic agents combined with immunotherapy regimens may have synergistic antitumor activity. After ALK TKI resistance, the combination of immunotherapy and anti-angiogenic agents may have greater clinical benefit than chemotherapy alone or in combination with anti-angiogenic agents. Preliminary data from several clinical trials exploring anti-angiogenic agents in combination with immune checkpoint inhibitors have been published Table 4.These results also led us to further consider whether inhibition of angiogenesis could be to increase the sensitivity of tumor cells to ICIs. Firstly, tumor angiogenesis induces immunosuppression in the body mainly by maintaining an acidic or hypoxic immunosuppressive tumor microenvironment, thereby inhibiting the maturation of dendritic cells and limiting T cell migration. In addition, studies have found that angiogenic factors  also have immunosuppressive functions, which to some extent inhibit the body’s killing of tumor cells . TherefoThe concept of vaccines for the treatment of cancer refers to the introduction of tumor associated antigens (TAAs) into the patient, thereby overcoming the immunosuppressive state of the body caused by the tumor and further inducing body humoral immunity and cellular immunity, thus achieving the purpose of inhibiting tumor growth . Vaccinein vivo and improving survival in mice, while the results of the study found that this ALK vaccine could double the survival of mice with ALK positive NSCLC and reduce the number of tumor cells by 60%  vaccines , 118. ALs by 60% , 121.in vivo in mice with ALK positive NSCLC in preclinical trials  annual meeting showed that the vaccine combination was highly effective in patients with low PD-L1 expression in the tumor and poor response to nivolumab monotherapy. Currently, phase II clinical trials of CIMAvax-EGF vaccine are well underway (NCT02955290). In addition, clinical trials of CIMAvax-EGF with Pembrolizumab (k drug) for first-line treatment in patients with advanced NSCLC with high expression of PD-L1 are also ongoing. In addition, TG4010 (a poxvirus expressing tumor antigen MUCI and IL-2), an oncolytic virus product developed by transgene, is currently under clinical investigation for non-small cell lung cancer in combination with chemotherapeutic agents , 126. MeChimeric antigen receptor (CAR)-T cells are a class of artificially developed specific therapeutic T cells whose main function is to specifically recognize specific antigens on the surface of tumor cells, thereby clearing tumor cells in the body. Recently, CAR-T therapy has become one of the effective treatments for hematological malignancies. Preliminary data from a study using car-t cell therapy in patients with advanced NSCLC showed a disease control rate of 55% . , a type of antitumor effect by injecting ex vivo expanded T lymphocytes into patients to improve the body, similar to CAR-T therapy, may become one of the options for patients with solid tumors in the future . NSCLC iSince the first report of this therapy in 1986 , tumor iCurrent studies of tumor infiltrating lymphocyte (TIL) therapy for NSCLC are limited, but the high mutational load of NSCLC itself and the high responsiveness to ICIS suggest that this class of cancer is a predictable treatment option for ACT. There was clinical experience in the 1990s with the use of TILs in the treatment of NSCLC . In thisRecent intensive studies in lung cancer found the feasibility of cNeT as an emerging therapy for NSCLC . Becausein vitro experiments  in head and neck cancer and trastuzumab (anti-HER2) in breast cancer, among others. Since the ALK receptor is mainly distributed on the cell surface, neutralizing antibodies can be used to treat ALK mutated related tumors. In addition, the expression of ALK related genes is mainly distributed in the nervous system, making anti ALK antibodies less harmful to normal tissues. Recently, research targeting anti ALK antibodies is constantly being explored, and some newly developed monoclonal antibodies against ALK have been confirmed to inhibit the growth of ALK positive tumor cells in eriments .Injection of a human single chain variable fragment (scFv) antibody against the ALK ligand-binding domain (LBD) in a mouse model has been studied, and ALK positive glioblastoma growth was found to be inhibited. Stylianou et al , demonstALK rearrangements are present in 5-6% of patients with NSCLC and confer high sensitivity to ALK TKIs. After ALK rearranged NSCLC patients become resistant to one or more ALK TKIs, the next treatment options include chemotherapy, immunotherapy, and a combination of chemotherapy and immunotherapy, among others. Many preclinical studies have found higher PD-L1 expression on tumor cells in patients with ALK rearranged NSCLC than on other types of lung cancer cells, but clinical trials of single agent immunotherapy have not yielded significant clinical benefit. There are studies that consider ALK rearranged NSCLC driving the organism to generate an immunosuppressive TME and thus cause tolerance to ICIs in such patients. To further explore the efficacy of immunotherapy in patients with ALK rearranged NSCLC, data from clinical trials combining immune checkpoints with treatment strategies such as ALK TKIs, chemotherapy and anti angiogenic agents are summarized. Most treatment regimens targeting immunotherapy in combination with ALK TKIs suffer from significant toxicities and low efficacy. The simultaneous discovery of potential toxic effects of certain tyrosine kinase inhibitors in patients with ALK rearrangement who had received previous immunotherapy provides evidence for the need for further refinement of genetic testing before treatment of patients with lung cancer. No significant clinical benefit was also found in the combination of immunotherapy combined with chemotherapy, but the combination of immunotherapy with antiangiogenic regimens may provide further prolongation of survival for patients after ALK TKI resistance.ALK directed vaccines convert an immunosuppressive TME into a highly reactive TME, and their significant efficacy has been demonstrated in preclinical studies, together with the discovery of synergistic antitumor effects of the vaccine in combination with chemotherapy or immunotherapy. Studies of combination regimens of vaccines still require validation in a large number of preclinical and clinical studies, and the case for sustained efficacy based on vaccine therapy requires further exploration. The efficacy of CAR-T cell therapy mainly depends on the specific recognition of tumor cell surface antigens and possible damage to surrounding tissues. ACT therapy as a kind of CAR-T cell therapy, infusing ex vivo expanded T cells into patients improves the body’s antitumor ability. The requirement of ACT therapy for the hypermutability of the tumor cells themselves and for hyperresponsiveness to immunotherapy, indicates the potential significance of ALK rearranged NSCLC to this therapy. In an attempt to further improve the survival prognosis of patients with ALK rearranged NSCLC lung cancer, monoclonal antibodies against ALK related proteins are in a pipeline and have demonstrated certain efficacy in preclinical studies.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.SSH provided the initial idea for this review. RBQ and YYY were in charge of data acquisition and drafting of the article. MS revised the article. All authors contributed to the article and approved the submitted version.