EGFR), n = 5; programmed death‐ligand 1 (PD‐L1), n = 6]. Twenty‐eight scenarios compared PDT‐guided PM with chemotherapy alone . Twenty‐five scenarios compared PDT‐guided PM with chemotherapy alone, while varying the PDT approach. Thirty‐four scenarios (53%) were cost‐effective, 28 (44%) were not cost‐effective, and two were marginal, dependent on their country’s WTP threshold. When PDT‐guided therapy was compared with a therapy‐for‐all patients approach, all scenarios (100%) proved cost‐effective. Seven of 37 studies had been structured appropriately to assess PDT‐PM cost‐effectiveness. Within these seven studies, all evaluated scenarios were cost‐effective. However, 81% of studies had been poorly designed. Our systematic analysis implies that more robust health economic evaluation could help identify additional approaches towards PDT cost‐effectiveness, underpinning value‐based care and enhanced outcomes for patients with NSCLC.Precision diagnostic testing (PDT) employs appropriate biomarkers to identify cancer patients that may optimally respond to precision medicine (PM) approaches, such as treatments with targeted agents and immuno‐oncology drugs. To date, there are no published systematic appraisals evaluating the cost‐effectiveness of PDT in non‐small‐cell lung cancer (NSCLC). To address this gap, we conducted Preferred Reporting Items for Systematic Reviews and Meta‐Analyses searches for the years 2009–2019. Consolidated Health Economic Evaluation Reporting Standards were employed to screen, assess and extract data. Employing base costs, life years gained or quality‐adjusted life years, as well as willingness‐to‐pay (WTP) threshold for each country, net monetary benefit was calculated to determine cost‐effectiveness of each intervention. Thirty‐seven studies (50%) were included for analysis; a further 37 (50%) were excluded, having failed population‐, intervention‐, comparator‐, outcomes‐ and study‐design criteria. Within the 37 studies included, we defined 64 scenarios. Eleven scenarios compared PDT‐guided PM with non‐guided therapy [epidermal growth factor receptor  patients who will best respond, increasing both quantity and quality of life. Our analysis demonstrates the inherent value of precision medicines for NSCLC patients in over half of cases. However, to build the health economic evidence base further, cost‐effectiveness analyses should be appropriately designed to maximise the potential of precision diagnostic testing. ALKanaplastic lymphoma kinaseARMSamplification refractory mutation systemASCOAmerican Society for Clinical OncologyAUD$Australian dollarsCAD$Canadian dollarsCEAcost‐effectiveness analysisCETcost‐effectiveness thresholdCHFSwiss francsCNChinaEGFRepidermal growth factor receptorFISHfluorescent in situ hybridizationHK$Hong Kong dollarsHRMhigh‐resolution meltICERincremental cost‐effectiveness ratioIHCimmunohistochemistryIOimmuno‐oncologyISPORInternational Society for Pharmacoeconomic Outcomes ResearchLYGlife year gainedMETmesenchymal epithelial transition factorMTSmultiplex targeted sequencingNGSnext‐generation sequencingNMBnet monetary benefitNSCLCnon‐small‐cell lung carcinomaOWSAone‐way sensitivity analysisPAPpatient access programmePD‐L1programmed death‐ligand 1PDTprecision diagnostic testPICOSpopulation, intervention, comparison, outcome, and study designPMprecision medicinePSAprobabilistic sensitivity analysisQALYquality‐adjusted life‐yearRCTrandomized controlled trialRETrearranged during transfectionROS1c‐ros oncogene 1S$Singapore dollarsT790MEGFR gatekeeper mutationTKItyrosine kinase inhibitorWTPwillingness‐to‐pay1The World Health Organization (WHO) lists lung cancer as the most common cancer and leading cause of cancer death  , 3. RelaEGFR; gefitinib, erlotinib, afatinib, dacomitinib and osimertinib), TKI against anaplastic lymphoma kinase , v‐raf murine sarcoma viral oncogene homolog B1 , c‐ros oncogene 1 , mesenchymal epithelial transition factor , rearranged during transfection proto‐oncogene , neurotrophic tropomyosin receptor kinase  and immuno‐oncology (IO) drugs . The American Society of Clinical Oncology (ASCO) and Ontario Health guidelines state that the 60% of stage IV NSCLC patients with actionable mutations  should be offered the corresponding precision medicine (PM) that targets these abnormalities, and the remaining 40% without driver mutations should be offered immunotherapy, dependent on programmed death‐ligand 1 (PD‐L1) tumour proportion score test results (((HRM test or HRMA test or sanger sequencing or pyrosequencing or high resolution) adj3 melt) or next generation sequencing).mp.  adj8 mutation)).mp. or ((genetic* adj test*) or ).tw. (immunohistochemistry or ihc).mp. biomarker.mp. adj (medic$ or treatment or therap$)).ti,ab,ot,hw.5 or 6 or 7 or 8 or 9 or 10 or 11.mp.(Anti‐EGFR therapy or EGFRi or Erlotinib or Tarceva or Afatinib or Gilotrif or Gefitinib or Iressa or Osimertinib or Tagrisso or Dacomitinib or Vizimpro).mp..mp.(Trametinib or Mekinist or Dabrafenib or Tafinlar).mp.(angiogenesis inhibitors or Bevacizumab or Avastin or Ramucirumab or Cyramza).mp.(Chemotherapy or Cisplatin or Carboplatin or Paclitaxel or Taxol or Abraxane or docetaxel or Taxotere or Gemcitabine or Gemzar or Vinorelbine or Navelbine or Etoposide or VP‐16 or Pemetrexed or Alimta).mp.surgery.mp. Radiotherapy.mp.or/13–20Costs.mp. and Cost‐Analysis/Cost‐Benefit Analysis/exp Models, Economic/Quality‐Adjusted Life Year*.mp.Economic Evaluation/Cost‐Effectiveness Analysis/Cost Utility Analyses/Statistical Model/(econom* or pharmacoeconomic* or pharmaco‐economic*).ti..tw.).tw..tw.(euroqol or eq5d or eq‐5d or hui or hui1 or hui2 or hui3).mp.) or (time adj2 trade) or (standard adj2 gamble)).mp.((cost* or economic*) adj2 model*).tw..ti,ab.(economic$ or cost or costs or costly or costing or costed or price or prices or pricing or priced or discount or discounts or discounted or discounting or ration$ or expenditure or expenditures or budget$ or afford$ or pharmacoeconomic or pharmaco‐economic$).ti,ab.exp economics, hospital/economics, medical/economics, nursing/economics, pharmaceutical/(expenditure$ not energy).ti,ab..ti,ab.budget$.ti,ab.(cba or cea or cua).ti,ab.exp "fees and charges"/(fee or fees or charge$ or preference$).tw..tw.exp Health Care Costs/).ti,ab. adj3 (money or monetary or life or lives or costs or cost$)).ti,ab..ti,ab.cost$.ti,ab.exp decision support techniques/markov$.ti,ab. or markov chains/monte carlo.ti,ab. or monte carlo method/).ti,ab. or decision tree/.ti,ab."deductibles and coinsurance"/exp Health expenditures/uncertain$.ti,ab. or uncertainty/.ti,ab. or quality of life/.ti,ab. or value of life/utilit$.tw.valu$.tw.exp hospitalization/.tw.or/22–684 and 12 and 21 and 69limit 70 to yr="2009 ‐ Current"Searched 1 January 2020.2.3Articles were screened for eligibility based on criteria listed in Table The integrity of each study was assessed according to a checklist developed by the ISPOR Consolidated Health Economic Evaluations Reporting Standards Task Force Report . This unn = 36) were removed. A total of 74 articles were screened for eligibility. After full text examination, 10 articles were either reviews or systematic reviews, which were retained for reference, whereas seven articles did not mention the terms life‐years gained (LYG), quality‐adjusted life‐years (QALY), or incremental cost‐effectiveness ratio (ICER). Seven other articles did not include cost‐effectiveness analysis (CEA), cost benefit analysis or cost utility analysis. On further examination, 12 were abstracts without enough detailed information, and one was an intervention without deployment of a PDT. In total, 37 eligible studies remained which involved economic evaluation of PDT for guiding therapeutic intervention in NSCLC.The Study Selection Workflow is outlined in Fig. 2.4excel. Extracted features included: author, year, country of study, NSCLC stage/advanced/not described, therapy, biomarker utilised, LYG, QALY, the current ICER (cost per LYG) and/or ICER (cost per QALY), willingness‐to‐pay (WTP) cost‐effectiveness threshold (CET) and net monetary benefit (NMB) statistic . We also extracted author, PM cost, PDT cost , perspective , modelling approach, time horizon (duration of therapy), discounting applied, one‐way sensitivity analysis (OWSA), probabilistic sensitivity analysis (PSA) and the trial upon which the economic evaluation was based. While most studies only listed one scenario of PDT intervention compared with standard of care (or another PDT), some studies listed as many as 10 different scenarios, where the scenario involved variation in the PDT, therapy or country. If there were insufficient data (e.g. abstract reports from conferences), we emailed the original authors for further details.We extracted empirical and methodological data and imported these data into Microsoft 2.5Data capture and quality analysis for each study of cost‐effectiveness were represented in the data extraction and as a narrative summary. Modelling techniques used in the different studies were compared and their robustness analysed.2.6Net monetary benefit was calculated in each instance where a PM‐guided by a PDT was compared with the same PM drug administered to all patients without PDT guidance.2.7In cases where more than one therapy and test combination were modelled, the reported ICER might not be compared to the base case, e.g. best supportive care (BSC) or LYG and QALY reported, but no corresponding ICER calculated. In these instances, we calculated the ICER based on reported costings and QALY for the PDT using the following formula:For the studies that compared PDT‐guided therapy with unselected PDT therapy for all‐comers and with chemotherapy, we conducted a sub‐analysis using the NMB  with the formula:The WTP CET employed for each scenario corresponded to that reported in the study; if more than one or a range of WTP CET were described, we conservatively chose the lowest. Additionally, if no WTP CET was disclosed, then the WTP CET from the same country in another captured study was employed, or 1× gross domestic product (GDP) per capita of that country was used.3The 37 studies were reported from Asia, Australia, Europe and North America, all of which were at least upper middle‐income countries. Publications spanned the period 2009–2019. Where a negative ICER was reported or calculated, it was always due to negative costs, not negative LYG or QALY. The reader should refer to the NMB statistic before drawing conclusions, as a negative value here will always determine that the intervention was not cost‐effective.3.13.1.1EGFR test is employed to guide gefitinib therapy, as opposed to an unselected approach (Table Four of five ICER were negative Table 2a), generated from increased QALY and less costly therapy; one of the ICER was positive , 19, 20.a, genera3.1.2)100,000 WTP CET (Table 2b)  to 4.24% . IHC tests were consistently the least expensive, while costs increased with the complexity of the test employed.The annual cost of each study’s PM was identified and this was employed as a denominator to assess the fraction of test cost relative to precision therapy Tables . The pro3.3The modelling approaches principally followed the Markov process  and probabilistic sensitivity analyses (PSA) conducted indicated in four of five cases that PDT employed to guide therapy were the dominant strategy in France, Singapore, South Korea and USA, demonstrating clinical benefit and cost‐effectiveness for the patient Table  16, 17,, 19, 20.3.4.2For immunotherapy guided by PD‐L1, the OWSA indicated that PDT deployment or patient’s health status had the greatest effect on the ICER, and the PSA determined that PD‐L1 testing increased cost‐effectiveness of the therapy in China, Switzerland and USA Table  21, 22], 2221, 23.4.3EGFR and the health status of the patient had the greatest impact on the ICER. The PSA determined in four of seven studies that in China (with patient access programmes), Germany and Japan, EGFR‐guided therapy was cost‐effective, whereas the Mexican, Thai and US studies were not  of these scenarios were deemed cost‐effective. However, only 11 of the 64 scenarios followed the correct analysis format to assess whether a PDT adds value to a PM approach. That is, only these 11 scenarios compared PM and PDT with PM administered to the patient cohort without prior use of the test to select patients. Of these, seven scenarios (63.6%) agreed with our hypothesis of PDT‐guided treatment conferring measurable increased benefit. Four scenarios presented conflicting results of data from Wan et al.;  we belieet al.; .EGFR‐directed therapy were greater in the Asian studies than in North American or European populations, which is to be expected, as the prevalence of EGFR mutations is greater in Asia. In 59%(24 of 41 cases), the EGFR‐guided therapy failed cost‐effectiveness criteria regardless of test type; this is also true for ALK testing in 38% (five of 13) of cases, and in 14% (two of 14) of PD‐L1 testing .Overall, the LYG or QALY gained for A number of the testing scenarios involving next‐generation sequencing (NGS) have difficultly capturing more than one actionable mutation with standard CEA, as current Markov or state transition models aggregate patient data into distinct health groups , neglecting heterogeneity amongst the patient cohort, and PSM is incapable of returning to PFS from a PD state, where in some cases there is a distinct possibility of a ‘cure’. Dynamic simulation modelling such as discrete event simulation (DES) has recently been suggested as a model which can track individual patient pathways, incorporating results, testing and consequential therapies .Our analyses strongly suggest that health economic evaluation should be performed routinely from the start of and alongside clinical trials. This is particularly true for precision oncology, where therapeutic costs are high and improved patient outcomes achieved through application of a relatively inexpensive PDT would be beneficial, both from a clinical and a health economic viewpoint. Previously, we have demonstrated a paucity of CEA studies for PM‐guided care in colorectal cancer; that same dearth of application of CEA is evident for NSCLC, with only seven of 37 studies (18.9%) adequately designed to analyse the cost‐effectiveness and value of PDT .4.1The principal strength of this systematic review is that we employ the NMB summary statistic rather than the ICER to assess cost effectiveness. NMB incorporates both costs and QALY at a WTP threshold particular to that country, allowing cost‐effectiveness to be easily captured, thus generating more robust data. Secondly, we demonstrate that while PM is a driver of costs, PDT are a driver of value. PDT, at a fraction of the cost of a precision therapy, add value beyond the therapy, by selecting patients who will accrue greater health benefits and reducing costs by excluding patients who will not benefit from a particular PM approach.Weaknesses of the data presented in this systematic review are that the majority of studies published are inappropriately structured to best assess effective PDT deployment in PM, which may reflect the lack of involvement of health economists and diagnostic stakeholders in setting the PM agenda.Secondly, the generalisability of the results of this study is difficult to ascertain, as WTP CET vary not only between but also within countries where such studies are performed. WHO proposes that it is reasonable to spend income to achieve a QALY that is equivalent to the GDP per capita of a country, a recommendation followed in the UK by the National Institute for Health and Clinical Excellence with its £30,000 WTP CET, but this is adapted for end‐of‐life disease such as metastatic NSCLC at £50,000, and modified again for small patient subgroups, a hallmark of PM, with values of £75,000 upwards , 45. SucThirdly, all these CEA are based on randomised controlled trials (RCT) which involve highly selected patient populations. For PM RCT, the small patient populations and more complex clinical pathways may increase uncertainty in CEA modelling results. Adding CEA of real‐world data as an adjunct to RCT data would improve confidence in a treatment’s effectiveness .Fourthly, the CEA do not capture capital costs (testing equipment), personnel and their training, and reporting tool costs.Fifthly, patient waiting times between test and therapy and the impact of first and potentially further surgical biopsies are not reported, two important aspects of PDT deployment. The turnaround times from sequential single gene testing to NGS is important in advanced NSCLC, where appropriate speed of test turnaround may be crucial to a patient’s survival (and likely QALY impact). These are not modelled in the studies described. Liquid biopsies also add to the speed of tumour profiling, with the additional bonus of sampling being relatively painless to the patient .5Over half of the scenarios analysed presented ICER below the WTP CET, suggesting a potential publication bias which can only be addressed by increased diligence and transparency in the health economics/precision oncology evaluation. Only seven of 37 CEA studies performed to assess the benefit of PM approaches in NSCLC care were appropriately designed to assess the value of combining PDT with PM, highlighting the need for greater emphasis on precise health economic analysis to inform value‐based patient care. Despite this, employing molecular tests to guide NSCLC therapy appears to be cost‐effective in the majority of cases. Thus, cost‐effective deployment of PDT can add substantial value to the PM approach well in excess of the cost of the test itself and should inform a more robust approach for future PM delivery for NSCLC patients.ML reports honoraria received from Pfizer, EMD Serono, Roche and Carnall Farrar for presentations unrelated to this work. ML also reports an unrestricted educational grant from Pfizer for activity unrelated to this work. None of the other authors have any conflicts of interest to declare.RH, PK, DF, DS and ML were involved in the conception and design of the study. RH, DF, DS and ML created the search strategy and performed the article inclusion. RH performed the data extraction. RH, DF, DS and ML performed the quality assessment. All authors contributed to data interpretation, critically revised the article, and approved the final version.Fig. S1. PRISMA flow diagramTable S1. Screening criteria and study design for systematic review.Table S2. CHEERS criteria and quality rating.Table S3a. Methodological characteristics and quality rating of TKI treatment guided by EGFR status versus TKI treatment for all patients.Table S3b. Methodological characteristics and quality rating of immunotherapy guided by PD‐L1 positivity versus immunotherapy for all patients.Table S3c. Methodological characteristics and quality rating of TKI treatment guided by EGFR or ALK status versus chemotherapy.Table S3d. Methodological characteristics and quality rating of assessment of immunotherapy guided by PD‐L1 positivity versus chemotherapy.Table S3e. Methodological characteristics and quality rating of treatment guided by genetic status using different testing scenarios.Click here for additional data file.


Nicotine can diversely affect neural activity and motor learning in animals. However, the impact of chronic nicotine on striatal activity in vivo and motor learning at long‐term sparse timescale remains unknown. Here, we demonstrate that chronic nicotine persistently suppresses the activity of striatal fast‐spiking parvalbumin interneurons, which mediate nicotine‐induced deficit in sparse motor learning. Six weeks of longitudinal in vivo single‐unit recording revealed that mice show reduced activity of fast‐spiking interneurons in the dorsal striatum during chronic nicotine exposure and withdrawal. The reduced firing of fast‐spiking interneurons was accompanied by spike broadening, diminished striatal delta oscillation power, and reduced sample entropy in local field potential. In addition, chronic nicotine withdrawal impaired motor learning with a weekly sparse training regimen but did not affect general locomotion and anxiety‐like behavior. Lastly, the excitatory DREADD hM3Dq‐mediated activation of striatal fast‐spiking parvalbumin interneurons reversed the chronic nicotine withdrawal‐induced deficit in sparse motor learning. Taken together, we identified that chronic nicotine withdrawal impairs sparse motor learning via disruption of activity in striatal fast‐spiking parvalbumin interneurons. These findings suggest that sparse motor learning paradigm can reveal the subtle effect of nicotine withdrawal on motor function and that striatal fast‐spiking parvalbumin interneurons are a neural substrate of nicotine's effect on motor learning. Chronic nicotine diminished the activity of fast‐spiking parvalbumin interneurons and degrades delta oscillation power as well as signal complexity of local field potentials in the dorsal striatum. Additionally, chronic nicotine impaired sparse motor skill learning specifically during nicotine withdrawal. Restoring the activity of striatal fast‐spiking parvalbumin interneurons rescued the chronic nicotine‐induced deficit in sparse motor skill learning. Specifically, we compared the in vivo activity of striatal medium spiny neurons (MSNs) and fast‐spiking interneurons (FSIs) before, during, and after chronic nicotine treatment. MSNs comprise ~95% of the striatal neuron population and are the output neurons of the striatum,Subsequently, we applied the chemogenetic approach to selectively modulate the activity of striatal fast‐spiking parvalbumin interneurons to examine their role in nicotine‐dependent alteration of motor learning. Here, we applied a sparse motor learning paradigm. The sparse training regimen renders the behavioral task more demanding, which has been shown to be more sensitive to subtle deficit in behavior.22.1tm1(cre)Arbr/J (PV‐Cre) heterozygous male mice  were group‐housed (two to four mice per cage) and handled for more than 1 week prior to the beginning of experiment. All mice were kept on a 12‐h reverse light/dark cycle (0800 lights off) with ad libitum access to food and water. All experiments took place during the dark phase. Animals were randomly assigned to each experimental group. All procedures regarding the use and handling of mice were conducted as approved by the Institutional Animal Care and Use Committee (IACUC) of the Korea Institute of Science and Technology (KIST).For in vivo single‐unit recording, 3‐month‐old C57BL/6J male mice  were single‐housed and extensively handled for more than 2 weeks before tetrode microdrive implantation surgery. For behavioral experiments, 2‐ to 3‐month‐old C57BL/6J male mice  or B6.129P2‐Pvalb2.2Mice were exposed to nicotine via subcutaneous implantation of osmotic mini‐pump . Briefly, mice were anesthetized with isoflurane . Nicotine solution was prepared immediately before osmotic pump implantation. (−)‐Nicotine ditartrate  was dissolved in physiological saline, and pH was adjusted to 7.4. Concentration was adjusted to deliver free‐base nicotine at 24 mg/kg/day for 2 weeks as previously described.Single‐unit recording and behavior tests were conducted over 6 weeks Figures , and S4.2.3Single units were recorded simultaneously from the right dorsal striatum  with four tetrodes. Tetrode was fabricated by twisting four strands of nichrome wire (RO800 0.0127 mm (0.0005″) 1/4 Hard PAC; Kanthal, FL, USA) together and heating to fuse the insulation. The tetrode tip was cut and gold plated to reduce impedance to 0.2–0.3 MΩ following a previous study.Single units were isolated via inspection of the 2D projections of waveform parameters and manually sorting the spikes in each cluster using custom software The firing rates and averaged spike waveform parameters of MSNs and FSIs were compared among three phases of recording sessions  Figure . As depiSpike waveforms were drawn by averaging the spike traces of units identified as either MSNs or FSIs across the three phases of recording sessions. For spike waveform analysis, we analyzed half‐width, peak‐to‐valley, spike amplitude, and amplitude ratio as in previous studies.In addition, the burst activity of MSNs were analyzed in parallel Figure . Burst a2.4r was used as the arbitrary threshold of similarity between two template sequences.Local field potential (LFP) recording was performed as described previously,2.5After the single‐unit recording was completed, mice were deeply anesthetized by intraperitoneal administration of Avertin  , and microlesions were made on the recording sites by passing an electrolytic current  through one channel of each tetrode. Subsequently, the whole brain was quickly isolated and freeze stored at −80°C. The frozen brain was coronally microdissected on a cryostat  to a thickness of 30 μm at −18°C. The brain sections were mounted on Superfrost Plus slide and air‐dried for ~10 min at room temperature. Then, the whole slide was sequentially incubated in xylene for 20 min, 100% ethanol for 5 min, 95% ethanol for 5 min, 70% ethanol for 5 min, distilled water for 5 s, cresyl violet  for 10 min, distilled water for 2 min, 70% ethanol for 5 min, 95% ethanol for 5 min, 100% ethanol for 5 min, and xylene for 20 min. Finally, the slide was mounted with Permount mounting medium mixed with xylene at 1:1 ratio. The sections were imaged under light microscopy Figure .2.6Mice were acclimated to the behavior test room for at least 30 min before the beginning of each experiment. The behavioral apparatus was wiped with 70% ethanol and distilled water before and between experimental sessions or blocks. The whole behavioral sessions were video recorded. As depicted in Figures 2.6.1A white open field arena (40 * 40 * 40 cm inner dimension) was used. Luminosity on the floor of the open field was adjusted to ~5 lux. Mice were placed in the open field and allowed to freely explore the arena for 30 min. The total distance moved and time spent in the center zone were analyzed using EthoVision XT 11.5 .2.6.2The luminosity on the floor of the light chamber was adjusted to ~350 lux. Mice were placed in the dark chamber; then, the door to the light chamber was opened, and mice were allowed to freely explore the chambers for 10 min. The time spent in the light chamber was measured.2.6.3Based on previous studies showing that the motor learning retention can be maintained for at least a week,Baseline indicates the mean latency to fall for an experimental group at the Baseline phase and PerformanceSession indicates the latency to fall for each mouse in the experimental group at one session of a phase .To quantitatively assess the within‐group, sparse training‐dependent improvement in motor skill, we analyzed motor learning index based on previous studies,2.712 GC/ml (Genome Copy/ml) for pAAV‐hSyn‐DIO‐mCherry and 3.70 * 1012 GC/ml for pAAV‐hSyn‐DIO‐hM3D(Gq)‐mCherry.pAAV‐hSyn‐DIO‐hM3D(Gq)‐mCherry  and pAAV‐hSyn‐DIO‐mCherry  were used for Cre‐dependent expression of an excitatory DREADD hM3Dq. Adeno‐associated virus (AAV) was packaged in the KIST Virus Facility . Virus titer was 9.86 * 102.8For intrastriatal microinfusion of AAV, mice were anesthetized by intraperitoneal administration of ketamine‐xylazine mixture  and appropriately positioned in a stereotaxic frame . Microinfusion was made into both hemispheres of the dorsal striatum at the following stereotaxic coordinates: AP, +0.7 mm; ML, ±1.9 mm from midline; and DV, −2.3 mm below the dura. The infusion was carried out by NE‐4000 Programmable 2 Channel Syringe Pump . The infusion volume was 0.2 μl, and the infusion rate was 0.1 μl/min. After infusion, the microneedle was held in position for 2 min.2.9N‐oxide (CNO) dihydrochloride (Tocris) was dissolved in physiological saline to 0.03 mg/ml. For activation of the excitatory DREADD hM3Dq, the CNO solution was injected through intraperitoneal administration route at a concentration of 0.3 mg/10 ml/kg at 30 min prior to the beginning of each behavioral session or transcardial perfusion.Clozapine 2.10Mice were deeply anesthetized by intraperitoneal administration of Avertin (250 mg/20 ml/kg). Then, transcardial perfusion was performed with 1× PBS followed by 4% paraformaldehyde (PFA) in 1× PBS. The whole brain was isolated, post‐fixed in 4% PFA for overnight at 4°C, dehydrated with 30% sucrose until submersion, embedded in OCT compound, and stored at −80°C until cryosectioning. OCT‐embedded brains were coronally microdissected on a cryostat to a thickness of 40 μm at −20°C. Free‐floating sections were rinsed in 1× PBS followed by 0.3% Triton X‐100 in 1× PBS (PBST). Washed sections were blocked with 10% normal donkey serum (NDS) in 1× PBST for 1 h, then the sections were incubated with rabbit anti‐c‐Fos (1:2000) , mouse anti‐parvalbumin (1:1000) , rabbit anti‐cleaved caspase‐3 (1:500) , or rabbit anti‐DsRed (1:500)  for 2 days (anti‐c‐Fos) or overnight in 1× PBST with 1% NDS at 4°C. Subsequently, the sections were washed and incubated with appropriate secondary antibody for 2 h at room temperature. Finally, the sections were washed and mounted with VECTASHIELD HardSet Antifade Mounting Medium with DAPI  according to the manufacturer's protocol. Images were taken with FluoView FV1000  and processed using FluoView software (Olympus); then, the images were visualized using Zen software  and ImageJ .2.11Dunn multiple comparison was applied because the sample distribution of spike properties was determined to be nonnormal .33.1To first examine the impact of chronic nicotine on the striatal activity in vivo, we recorded the single‐unit activity of striatal neurons throughout 6 weeks of weekly longitudinal single‐unit recording sessions. Briefly, we implanted tetrodes onto the dorsal striatum of five mice Figure  and expon = 85, 115, and 109 for Baseline, Exposure, and Withdrawal, respectively) but significantly and persistently diminished the firing rate of FSIs during both nicotine exposure and withdrawal phases  = 10.85; *p = 0.0212 for Baseline vs. Exposure, **p = 0.0055 for Baseline vs. Withdrawal).To identify the long‐term effect of chronic nicotine on the activity of striatal MSNs and FSIs, we compared the change in firing rate across three phases of recording sessions  Figure . QuantitH(2) = 8.400; *p = 0.0413 for Baseline vs. Exposure, *p = 0.0233 for Baseline vs. Withdrawal), whereas substantially broadened the half‐width (H(2) = 17.33; ***p = 0.0007 for Baseline vs. Exposure, ***p = 0.0004 for Baseline vs. Withdrawal) and peak‐to‐valley (H(2) = 14.49; **p = 0.0011 for Baseline vs. Exposure, **p = 0.0029 for Baseline vs. Withdrawal) of the FSIs.To subsequently identify whether the impact of chronic nicotine extends to the intrinsic properties of striatal neurons, we analyzed spike waveform parameters of striatal MSNs and FSIs across three phases of recording sessions. Through visual inspection, we noticed that the averaged spike waveforms of MSNs display a subtle decrease in valley amplitude during both nicotine exposure and withdrawal Figure . Meanwhin = 4 per group).Previous studies have well‐established that the striatal FSIs are marked by parvalbumin immunoreactivity.F = 149.7; ****p < 0.0001 for Baseline vs. Exposure, **p = 0.0015 for Baseline vs. Withdrawal) but did not affect the power of other frequency bands. In addition, chronic nicotine also reduced the sample entropy (SampEn)  = 76.03; **p = 0.0067 for Baseline vs. Exposure, **p = 0.0029 for Baseline vs. Withdrawal at r = 0.1).Thereafter, we carried out LFP analyses to identify the effect of chronic nicotine on striatal field activity. LFP power analysis revealed that chronic nicotine exposure and withdrawal reduced the normalized PSD of delta oscillation  = 4.662; #p = 0.0195 for Control vs. Nicotine during Withdrawal). Interestingly, sparse motor learning was impaired only during nicotine withdrawal and not during nicotine exposure, although there was a trend towards deficit .First, we found that chronic nicotine did not significantly affect general locomotion and anxiety‐like behaviors in the open field and light–dark transition tests Figure . HoweverF = 16.20; ***p = 0.0006, ****p < 0.0001), but the sparse learning‐dependent improvement in motor performance was hindered by chronic nicotine  at 30 min prior to the beginning of each behavioral experiment.In association with the data from single‐unit recording, we suspected that the nicotine‐induced reduction of activity in striatal fast‐spiking parvalbumin interneurons could be the antecedent of the deficit in sparse motor learning. Therefore, to assess the role of striatal fast‐spiking parvalbumin interneurons in nicotine‐induced motor learning deficit, we adopted the chemogenetic approach to reverse the nicotine‐induced reduction of firing in striatal fast‐spiking parvalbumin interneurons Figure . To thisn = 18, 18, and 16 for Control, Nicotine, and Nicotine + PVhM3Dq, respectively; F = 2.984; #p = 0.0107 for Control vs. Nicotine and #p = 0.0420 for Nicotine vs. Nicotine + PVhM3Dq).First, the distance moved or time spent in the center zone in the open field test were unaffected by chemogenetic activation of striatal parvalbumin interneurons in nicotine‐exposed PV‐Cre mice Figure . Next, CF = 31.49; ****p < 0.0001), but chronic nicotine led to only marginal increase in the learning index . Also, the chronic nicotine‐induced reduction of motor learning index was reversed by CNO‐induced activation of striatal parvalbumin interneurons .In accordance, the motor learning index gradually and substantially increased through sparse motor learning in PV‐Cre mice . We found that neither CNO nor chronic nicotine with CNO affected the distance moved and time spent in the center zone in the open field test  = 5.301; ##p = 0.0096 for Control vs. Nicotine + CNO, ###p = 0.0004 for CNO vs. Nicotine + CNO).Lastly, previous studies have shown that CNO alone could affect behavior possibly through back‐conversion of CNO to clozapine and subsequent interaction between signaling from DREADD with signaling from endogenous receptors associated with clozapine.t Figure . In addiF = 26.44; **p = 0.0088 for Baseline vs. Exposure, **p = 0.0066 for Exposure vs. Withdrawal, ****p < 0.0001 for Baseline vs. Withdrawal) whereas the chronic nicotine‐dependent reduction in the learning index was unaffected by CNO  an extension of subtle motor deficit from nicotine exposure or (2) a withdrawal‐specific deficit that was newly arisen after nicotine exposure. Supporting the former idea, the subtle reduction in motor performance observed during nicotine exposure implies that the impaired motor learning may have already been in place during exposure and lasted throughout withdrawal. However, the slope of learning was positive from baseline to exposure, whereas the slope of learning reached towards 0 from exposure to withdrawal. This suggests that motor learning was partially intact during nicotine exposure but was evidently absent during nicotine withdrawal. Collectively, we support the latter view that an exposure‐independent, withdrawal‐specific motor deficit caused the chronic nicotine‐dependent impairment in sparse motor learning.In our study, anxiety‐like behavior was unaffected by chronic nicotine treatment. This finding is consistent with previous reports showing that chronic administration of nicotine does not affect anxiety‐like behavior in male mice.Lastly, the neurobehavioral difference between nicotine exposure and withdrawal was not observed in our study. Because the methods used in our study were limited in number and diversity, other neurophysiological or behavioral analyses could reveal the potential difference. In accordance, further research topics of interest include the longitudinal impact of chronic nicotine on other striatal interneurons/cell types and other dorsal striatum‐associated behaviors . Moreover, the differential molecular alterations induced by nicotine exposure and withdrawal in the dorsal striatum would be another interesting topic to explore in the future.4.3Only the trait phenotype was examined in the single‐unit recording experiment. The difficulty of single‐unit recording during rotarod test precluded us from examining the state phenotype, that is, neural activity during behavior. Nonetheless, information from the state phenotype is valuable for understanding the impact of chronic nicotine on behavior. Also, striatal cholinergic interneurons were not reliably identified and hence excluded from subsequent analysis. Previous studies found that striatal cholinergic interneurons are an important cellular substrate of motor function and dysfunction.A main focus in our study was to reveal whether the striatal fast‐spiking parvalbumin interneurons are involved in nicotine‐induced motor deficit. In doing so, we have not explored other relevant points: (1) The neurophysiological consequences arising from chemogenetic activation of striatal fast‐spiking parvalbumin interneurons and (2) the intermediate mechanism by which the restoration of activity in striatal fast‐spiking parvalbumin interneurons reverses the behavioral effect of chronic nicotine. Without a thorough understanding of the impact of striatal fast‐spiking parvalbumin interneurons on nicotine‐associated behaviors and striatal activity, it would be premature to attribute the striatal fast‐spiking parvalbumin interneurons as the sole mediator of nicotine‐induced motor deficit.Most importantly, the absence of relevant experimental groups in behavioral data limits us to make conservative interpretations. First, we did not use female animals in our study, precluding us from generalizing the data to the overall population. Although nicotine discrimination and seeking behaviors does not seem to differ between males and females,5We employed a sparse motor learning paradigm to identify that chronic nicotine withdrawal leads to impairment in motor learning. We revealed through 6 weeks of longitudinal in vivo single‐unit recording and chemogenetic approach that the striatal fast‐spiking parvalbumin interneurons are a neural substrate of nicotine withdrawal‐induced motor learning deficit. Our data suggest that striatal fast‐spiking parvalbumin interneurons mediate the behavioral effect of nicotine and sparse motor learning, the notions of which may be extended to the research for nicotine dependence and movement disorders.The authors declare no conflict of interest.H‐II supervised the project. BK designed and performed experiments. BK and H‐II analyzed the data and wrote the paper. All authors have critically reviewed content and approved final version submitted for publication.Table S1. Normality analysis for the spike data from tetrode single‐unit recording. Shapiro–Wilk normality test revealed that the spike data does not follow normal distribution. Therefore, the spike data was statistically analyzed with a nonparametric test method. MSN indicates medium spiny neurons, FSI indicates fast‐spiking interneurons, and IBFR indicates intraburst firing rate.Click here for additional data file.Figure S1. Chronic nicotine does not alter the burst firing properties of medium spiny neurons. (A) The burst rate, (B) intraburst firing rate (IBFR), and (C) burst duration of putative striatal medium spiny neurons (MSN) were unchanged by chronic nicotine treatment in vivo. n = 85, 115, and 109 for Baseline, Exposure, and Withdrawal, respectively.Click here for additional data file.Figure S2. Chronic nicotine withdrawal does not lead to loss of the striatal parvalbumin interneurons. (A) Cleaved caspase‐3 (Casp‐3), a marker for neuronal cell death, was not induced in neither the striatal parvalbumin (PV) interneurons nor any other cells after 2 weeks of withdrawal from chronic nicotine exposure. (B) Striatal PV + cell density (cells/mm3) was not affected by chronic nicotine withdrawal. n = 4/group.Click here for additional data file.Figure S3. hM3Dq‐mediated activation of striatal parvalbumin interneurons. (A) Immunohistochemical analysis of c‐Fos expression co‐localized to the mCherry  signal. (B) The excitatory DREADD hM3Dq‐mediated, clozapine N‐oxide (CNO)‐induced increase in the activation of striatal parvalbumin (PV) interneurons verified by c‐Fos expression co‐localized to the PV signal (c‐Fos+/PV+) (*p = 0.0372). n = 5/group.Click here for additional data file.Figure S4. Clozapine N‐oxide does not affect sparse motor learning. (A) Neither clozapine N‐oxide  nor chronic nicotine with CNO  significantly altered the distance moved in open field test compared to the control . (B) Neither CNO nor chronic nicotine affected the time spent in the center zone in open field test. (C) Chronic nicotine decreased the latency to fall in the rotarod during withdrawal phase irrespective of the CNO treatment . (D) Mice exhibited sparse training‐dependent increase in motor learning index  irrespective of the CNO treatment . On the other hand, chronic nicotine attenuated the gradual increase in motor learning index irrespective of the CNO treatment (Nicotine+CNO group). B indicates Baseline, E indicates Exposure, and W indicates Withdrawal phase.Click here for additional data file.


These patients also had significantly higher proportions of hypertension (p = 0.006) and diabetes (p = 0.049) than the non‐NAFLD cases. No significant differences existed in the severity, mortality, viral shedding time and length of hospital stay between patients with or without NAFLD in the sample population. However, subgroup analyses found that in patients with normal body mass index (BMI), NAFLD sufferers were more likely to experience a severe event . Kaplan‐Meier curve (log‐rank p = 0.017) and Cox regression  analyses confirmed that before and after adjusting for gender, age and comorbidities, NAFLD patients with normal BMI had a higher incidence of suffering severe events. People with NAFLD may have a higher proportion of COVID‐19. NAFLD may be correlated with the severity of COVID‐19 patients in the normal BMI group.This study aims to evaluate the effect of non‐alcoholic fatty liver disease (NAFLD) on the susceptibility and consequences of coronavirus disease 2019 (COVID‐19). We retrospectively collected data from 218 adult COVID‐19 patients who showed no evidence of excessive alcohol consumption and underwent abdominal ultrasound examinations. Of these patients, 39.4% patients had been diagnosed with NAFLD, which indicates a much higher prevalence of NAFLD than that reported in the general population. Significantly elevated white blood cell count ( We retrospectively collected data from a cohort of subjects who had laboratory‐confirmed COVID‐19 and were patients in the Public Health Treatment Center of Changsha, China, before 14 March 2020. Fatty liver was diagnosed based on the ultrasound parameters  by abdominal ultrasonography with a 3.5‐MHz transducer . Ultrasonography was performed by the experienced radiologists for medical indications in the Public Health Treatment Center of Changsha, China. We used the following criteria to diagnose NAFLD: (1) The presence of fatty liver; (2) The absence of excessive alcohol consumption .2.2Two authors carefully collected and reviewed the individual medical records of the patients. Detailed information on demographic data, body mass index (BMI), underlying comorbidities, medical history, symptoms, laboratory parameters, chest computed tomographic (CT) scans and outcomes were recorded. For BMI data, the medical records of 18 patients did not include height and weight.2.3The primary endpoint of this study was a severe event, which was defined using the following criteria: (1) respiratory rate ≥30 breaths/min; (2) oxygen saturation <93%; (3) PaO2/FiO2 ≤ 300 mmHg; (4) lung lesions progressed to greater than 50% within 24–48 hours; (5) mechanical ventilation was implemented; (6) shock; (7) combined with other organ failures, required intensive care.2.4Because the continuous variables in this study were non‐normal distributed, we used the Fisher's exact test (χ2 test) and Mann‐Whitney test to compare the differences between the categorical variables and the continuous variables, separately. The Kaplan‐Meier (KM) method and the log‐rank test were applied to assess the association between NAFLD and the outcomes. Cox regression was conducted to further evaluate the effect of NAFLD after adjusting for other risk factors. All analyses were performed using IBM SPSS Statistics version 26.0 software.32 [18.7–37.2] vs 21.8 kg/m2 [11.7–29.3], p = 0.000) and higher proportions of hypertension  and diabetes . However, no significant differences were found in other comorbidities (including cardiovascular disease and chronic liver disease) and common COVID‐19 symptoms . Two of the patients (1.5%) died, while the remaining 216 patients were discharged before 14 March 2020 , alanine aminotransferase , aspartate aminotransferase , creatine kinase  and C reactive protein  were detected in the patients with NAFLD than in those without NAFLD. Meanwhile, the proportions of procalcitonin positive subjects  and the incidence of ground‐glass opacity obtained from chest CTs  were significantly higher in the NAFLD group than in the non‐NAFLD group (Table p = 0.018), low‐density lipoprotein (LDL) cholesterol , high‐density lipoprotein (HDL) cholesterol  and triglyceride , virus shedding time , length of hospital stay  and mortality .With regard to the outcomes, there was no significant difference between NAFLD and non‐NAFLD patients, including the severe events , NAFLD patients were more likely to develop severe events than those without NAFLD . However, no significant differences were found in patients with low (< 18.5 kg/m2) or high (≥ 24 kg/m2) BMI values (Table p = 0.000), aspartate aminotransferase , total bilirubin , C reactive protein  and triglyceride . Conversely, they had slightly lower HDL cholesterol levels  than those without NAFLD , those with NAFLD had a higher incidence of severe events , NAFLD was associated with an increased risk of severe disease. However, we did not find similar results in patients with higher and lower BMI, which may be related to their different metabolic states. This is an interesting result, but well‐designed studies with larger sample sizes are required to investigate this assumption.The susceptibility to COVID‐19 of NAFLD sufferers increases linearly with BMI. It increases 5‐ to 10‐fold in obese patients and by a factor of 10‐ to 14‐fold in the morbidly obese.2), due to the small number of obese patients (BMI ≥28 kg/m2). Finally, as this study was retrospective, data regarding abdominal adiposity was not available. Therefore, well‐designed studies with larger sample sizes are still needed to more clearly demonstrate the association between NAFLD and the outcomes of COVID‐19.This study contains several limitations. Firstly, we analysed the effect of NAFLD on the susceptibility and outcome of COVID‐19 in one of the nearest provincial capitals to Wuhan, but we did not have the data on the prevalence of NAFLD in Changsha, China. Also, in most of the subgroups that were created according to the common comorbidities, no significant differences were found. However, the results did show certain trends. Univariate analysis indicated that non‐elderly NAFLD patients had a higher incidence of severe events. Nevertheless, after adjusting for gender, hypertension and BMI, the difference became statistically insignificant. This may be because of the small sample size and the high incidence of comorbidities. Another limitation is that in the subgroup analysis on BMI, we only divided our patients into three groups. Overweight and obese patients were pooled together in one group ; Data curation ; Formal analysis ; Investigation ; Methodology ; Project administration ; Resources ; Writing‐original draft ; Writing‐review & editing . Shangjie Wu: Funding acquisition ; Investigation ; Supervision ; Validation ; Writing‐review & editing . Chenfang Wu: Data curation ; Investigation ; Supervision ; Writing‐review & editing . Quan Zhang: Data curation ; Formal analysis ; Investigation ; Methodology ; Resources . Fang Wu: Formal analysis ; Investigation ; Supervision ; Writing‐review & editing . Bo Yu: Conceptualization ; Data curation ; Methodology . Siye Zhang: Data curation ; Formal analysis ; Methodology . Chao Wu: Data curation ; Formal analysis ; Writing‐original draft . Guobao Wu: Supervision ; Writing‐review & editing . Yanjun Zhong: Conceptualization ; Data curation ; Formal analysis ; Funding acquisition ; Investigation ; Methodology ; Project administration ; Resources ; Supervision ; Validation ; Visualization ; Writing‐original draft ; Writing‐review & editing .Fig S1Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.


Studies indicate that uninterrupted anticoagulation (UA) is superior to interrupted anticoagulation (IA) in the periprocedural period during catheter ablation of atrial fibrillation. Still IA is followed in many centers considering the bleeding risk. This meta‐analysis compares interrupted and uninterrupted direct oral anticoagulation during catheter ablation of atrial fibrillation.A systematic search into PubMed, EMBASE, and the Cochrane databases was performed and five studies were selected that directly compared IA vs UA before ablation and reported procedural outcomes, embolic, and bleeding events. The primary outcome of the study was major adverse cerebro‐cardiovascular events.P = .006), whereas mean ACT was similar between the two groups (P = .19). Total heparin dose needed was significantly higher with IA . Mean procedure time did not vary between groups (P = .81). Overall complication rates were low, with similar major adverse cerebro‐cardiovascular event (P = .40) and total bleeding (P = .55) rates between groups. Silent cerebral events (SCEs) were significantly more frequent with IA . Rates of major bleeding, minor bleeding, pericardial effusion, cardiac tamponade, and puncture complications were similar between groups.The meta‐analysis included 840 patients with UA and 938 patients with IA. Median follow‐up was 30 days. Activated clotting time (ACT) before first heparin bolus was significantly longer with UA (UA during atrial fibrillation ablation has similar bleeding event rates, procedural times, and mean ACTs as IA, with fewer SCEs. This meta‐analysis compares interrupted and uninterrupted direct oral anticoagulation during catheter ablation of atrial fibrillation. Uninterrupted anticoagulation during atrial fibrillation ablation has similar bleeding event rates, procedural times, and mean activated clotting times as interrupted anticoagulation, with fewer silent cerebral events. Three physician reviewers  queried PubMed, EMBASE, and the Cochrane Central Register of Controlled Trials (CENTRAL) databases for published literature; search terms were “atrial fibrillation,” “catheter ablation,” “radiofrequency ablation,” “cryoballoon,” “hot balloon,” “uninterrupted,” “interrupted,” “novel oral anticoagulants,” “direct oral anticoagulants,” “dabigatran,” “rivaroxaban,” “apixaban,” “edoxaban,” “stroke,” “silent cerebral events,” and combinations of these keywords. Additional literature was sought by searching the references of eligible articles. Any inter‐reviewer discrepancies were resolved by a fourth reviewer (IBR).2.2For the qualitative synthesis of the meta‐analysis, we selected studies that (a) directly compared uninterrupted anticoagulation (UA) vs interrupted anticoagulation (IA) with a DOAC regimen before catheter ablation of AF and (b) provided procedural outcomes and embolic and bleeding events. Studies that involved both UA and IA with DOACs but did not report comparative outcome data for each regimen were excluded from the quantitative meta‐analysis. Single‐arm studies, case reports, case series, and cohort studies that had < 10 participants or that did not present adequate safety or efficacy outcome data also were excluded. See eFigure 1 in the Online 2.32DS2‐VASc  score, HAS‐BLED  score, left ventricular ejection fraction (LVEF), left atrium diameter, creatinine clearance, associated antiplatelet drugs, dimerized plasmin fragment D (D‐dimer) and brain natriuretic peptide levels, and presence of paroxysmal AF, coronary artery disease, chronic kidney disease, or structural heart disease. Procedural outcomes included procedure time, activated clotting time (ACT), heparin dose, cardioversion, and use of protamine. Efficacy outcomes included embolic events and silent cerebral events (SCEs). Safety outcomes included major bleeding events  and minor bleeding events .Baseline characteristics and safety and efficacy outcome data were extracted from each of the selected studies and entered into a Microsoft Excel spreadsheet by authors DK, AM, and SS. Baseline characteristics included DOAC regimen, number of participants, maximum follow‐up duration, age, sex, CHA2.4The primary outcome of the study was major adverse cerebro‐cardiovascular events (MACCVEs), which was a composite of stroke or TIA and major bleeding, total bleeding (composite of major and minor bleedings), and SCE. The secondary outcomes were cerebral embolic stroke or TIA, major and minor bleeding, total pericardial effusion, cardiac tamponade, and total puncture complications .2.52, and publication bias was assessed by funnel plot.To compare the safety and efficacy outcomes in the UA and IA groups, we used hypergeometric‐normal modeling to approximate the exact likelihood, as the number of events in each study was small relative to group size and included many zero events. To negate the small study effect, we calculated logarithmic odds ratios (log ORs) with 95% CIs and then used R software3Five studies with a total of 840 UA patients and 938 IA patients were included in the meta‐analysis; of these, three were randomized trials,3.12DS2‐VASc score, and the number of participants who had paroxysmal AF, had received antiplatelet drugs, or had structural heart disease were similar in both UA and IA groups across all studies. Maximum left atrial diameter, LVEF, creatinine clearance, and D‐dimer and brain natriuretic peptide levels did not vary significantly between the UA and IA groups.The various anticoagulant regimens are described in Table 3.2P = .003; I2, 88%). ACT before first heparin bolus was significantly longer in the UA group . No significant differences between the UA and IA groups were found for mean procedure time , mean ACT , maximum ACT , or minimum ACT . Protamine use was marginally higher in the UA group, but the difference not statistically significant , as shown in eFigure 2 in the Figure 3.3Clinical outcomes across the studies are described in eTable 2 in the 3.3.1P = .40; I2, 0%) or total bleeding . SCEs were significantly more frequent in the IA group .The UA and IA groups did not differ significantly in terms of MACCVE . Major and minor bleeding were also similar between the groups , as were total pericardial effusion (P = .67), cardiac tamponade (P = .73), and total puncture complications .There was no significant difference in stroke or TIA incidence between the UA and IA groups  study4.2The trials described above used direct anticoagulants that have important differences in pharmacodynamics and dosing, and they also used different protocols, resulting in heterogeneity. The two studies using a once‐daily DOAC shifted the last anticoagulant dose to the night before the procedure. In VENTURE‐AF, the last dose of rivaroxaban was administered predominantly on the evening before the procedure. Patients randomized to uninterrupted edoxaban in the ELIMINATE‐AF trial also took their scheduled doses in the evening.4.3Practical Guide on the Use of Non‐Vitamin K Antagonist Oral Anticoagulants in Patients with Atrial Fibrillation recommend continuation of oral anticoagulation with vitamin K antagonists or DOACs among patients undergoing AF ablation procedures.Multiple guidelines, international consensus statements, and, most recently, the European Heart Rhythm Association's 4.44.4.1Reynolds et al4.4.2The incidence of periprocedural thromboembolism in patients with AF undergoing ablation ranges from 0.9% to 5% and depends on the diagnostic modality.2DS2‐VASc score in the UA group and by intraprocedural cardioversion and procedure time in the IA group. Overall, SCE was significantly more frequent in the IA group (P < .005).Müller et al4.4.3Reynolds et al4.4.4MACCVE is a novel composite endpoint, we looked into, which comprised of major bleeding events as well as thrombotic events. In our meta‐analysis, MACCVE did not differ significantly between the UA and IA groups. Although SCE was noted more in relation to interrupted DOACs, the overall outcomes were comparable between the two groups which suggest that even with uninterrupted periprocedural anticoagulation, patients can be discharged safely from hospital following AF ablation on the same day.4.5To date, the clinical relevance of SCE remains unclear. Some data suggest that SCE is associated with cognitive impairment occurring after an AF ablation procedure.5First, we were able to include only five studies, two of which were observational trials. Second, the overall follow‐up duration was less. Third, there was considerable difference in the periprocedural anticoagulation regime across the studies. Fourth, subgroup analyses  could not be done because of lack of data since the data are heterogeneous and in consequence subgroups are small. Moreover, two of the five studies did not report kidney function (GFR), while this is relevant especially with DOACS. The presentation of atrial fibrillation differed significantly between the studies , which implies that the patients were affected by persistent or permanent AF. This may have an impact on rhythm variability, which may play a role in developing silent cerebral ischemia. The observational study by Nakamura6Compared with interrupted DOAC therapy, uninterrupted DOACs during AF ablation were associated with similar bleeding events and similar procedural times but lower rates of SCE, despite achieving a similar mean ACT. Further research is needed for risk stratification of the various DOAC regimens, understanding the predictors of SCE, and long‐term follow‐up of patients with SCE. On the basis of the information available thus far, we recommend truly uninterrupted DOAC treatment at the time of AF ablation.7Ethics approval and consent to participate‐ Not applicableConsent for publication‐ not applicableCompeting interests‐ noneFunding‐ noneAuthor contributions‐ IBR, DK, AM, PK, SKS, AA,JB, JJ, and AKConception and design: IBR, DK, PK, AK, and STAnalysis and interpretation of data: IBR, DK, AM, PK, SKS, and STDrafting of the manuscript or revising it critically for important intellectual content: IBR, DK, AM, PK, SKS, AA, JB, JJ, and AKFinal approval of the manuscript: IBR, DK, AM, PK, SKS, AA, JB, JJ, and AKAcknowledgments: Jeanie F. Woodruff, BS, ELS, contributed to the editing of this manuscriptSupporting data are available and can be accessed from the corresponding authorDisclosure statement‐ No conflict of interest to be declaredSupplementary MaterialClick here for additional data file.


Numerous effective HIV prevention options exist, including behaviour change interventions, condom promotion and biomedical interventions, like voluntary medical male circumcision and pre‐exposure prophylaxis. However, populations at risk of HIV also face overlapping vulnerabilities to common mental disorders and severe mental illness. Mental health status can affect engagement in HIV risk behaviours and HIV prevention programmes. We conducted a narrative review of the literature on HIV prevention among key populations and other groups vulnerable to HIV infection to understand the relationship between mental health conditions and HIV prevention outcomes and summarize existing evidence on integrated approaches to HIV prevention and mental healthcare.We searched five databases for studies published from January 2015 to August 2020, focused on HIV prevention and mental health conditions among key populations and individuals with serious mental illness. Studies were included if they evaluated an HIV prevention intervention or assessed correlates of HIV risk reduction and included assessment of mental health conditions or a mental health intervention.We identified 50 studies meeting our inclusion criteria, of which 26 were randomized controlled trials or other experimental designs of an HIV prevention intervention with or without a mental health component. Behaviour change interventions were the most common HIV prevention approach. A majority of studies recruited men who have sex with men and adolescents. Two studies provided distinct approaches to integrated HIV prevention and mental health service delivery. Overall, a majority of included studies showed that symptoms of mental disorder or distress are associated with HIV prevention outcomes . In addition, several studies conducted among groups at high risk of poor mental health found that integrating a mental health component into a behaviour change intervention or linking mental health services to combination prevention activities significantly reduced risk behaviour and mental distress and improved access to mental healthcare.Evidence suggests that mental health conditions are associated with poorer HIV prevention outcomes, and tailored integrated approaches are urgently needed to address overlapping vulnerabilities among key populations and other individuals at risk. Over 60% of new HIV infections occur among key populations and their sexual partners, including gay men and other men who have sex with men (MSM), female sex workers (FSW), transgender women (TGW) (primarily), people who inject drugs (PWID), and prisoners and other incarcerated people , universAmong key populations and other vulnerable groups, the same inequities that drive HIV infection also increase the risk for mental health conditions . Social Vulnerability to HIV and mental health conditions also intersect in adolescence, a sensitive period of neuropsychological and social development during which adolescents seek greater autonomy, takes risks and initiate sexual activity, with gender‐specific consequences , 16. GloLastly, people with SMI experience specific challenges related to living with symptoms that can be disabling, in addition to social, economic and gender‐specific vulnerabilities , 26, 27.Few studies have examined HIV prevention and mental health among HIV‐negative key populations and vulnerable groups eligible for combination prevention, condom promotion, PrEP and other HIV prevention services. HIV prevention programmes providing behavioural counselling and PrEP interventions have found that young key populations have a high prevalence of depressive, anxiety and traumatic stress symptoms , 31, 32.From 2014 to 2016, several reviews reported the evidence on the need for and results of HIV prevention interventions focusing on people with SMI , 36, 37.A synthesis of more recent literature on the range of mental health conditions (from stress to SMI) is needed to understand the latest evidence on HIV prevention interventions and their outcomes for people managing or at risk for mental health conditions. We conducted a narrative review of global research on mental health and HIV prevention among adolescents and young women, key populations and people with SMI. Our primary aim was to understand how symptoms of mental health conditions influence HIV prevention intervention engagement and outcomes among these groups. We also reviewed evidence of integrated approaches to HIV prevention and the management of mental health conditions. We organize our findings around two questions: 1) what do we know about the relationship between mental health and HIV prevention or risk behaviours? 2) what do we know about interventions that address mental health in the context of HIV prevention?2We conducted a structured narrative review of the literature in peer‐reviewed journals to examine the relationships between HIV prevention, risk reduction and mental health conditions.2.1Two authors worked with a university informationist to select search terms and determine filters for five databases. We searched PubMed, Web of Science, CINAHL, PsychINFO and EMBASE for English‐language studies published between 1 January 2010 and 25 August 2020. We constructed search terms on HIV prevention; interventions, services or programmes; and mental disorders. We ran these base search terms with search terms for each of the three populations of interest: (1) adolescents (boys and girls) and young women, (2) people with SMI and (3) UNAIDS‐defined key populations . (See A2.2Studies were eligible if they met the following criteria: (1) assessed correlates of HIV risk reduction activities or evaluated an HIV prevention intervention; (2) included assessment of mental health conditions, cognitive processes or tailored an intervention for people with mental disorders. We included studies related to the use of structural and behavioural HIV risk reduction approaches as well as biomedical prevention interventions. We excluded qualitative studies. Given the scope of the literature on drug use and HIV prevention, which merits its own review, we eliminated studies focused on harm reduction and prevention or treatment of substance use disorders if there was no mental disorder data. We also eliminated studies exclusively focused on HIV treatment as prevention for this review. Although treatment as prevention studies have the ultimate goal of preventing HIV transmission, they tend to focus on populations living with HIV who may have distinct HIV care and mental health needs compared with at‐risk, HIV‐negative populations. Eligible studies reported the outcomes of HIV prevention trials, cross‐sectional and longitudinal analyses or quasi‐experimental studies.2.3We restricted our review of papers to January 2015 to August 2020. There were overlapping topical reviews of the literature prior to 2015 (e.g. for people with SMI). Given the scope of prevention research publications and the rapid evolution of HIV prevention science, the criterion of a 5‐year period of review targets current research most relevant to practitioners and researchers. However, if studies from this time period were sub‐studies, secondary analyses or otherwise related to a primary trial that met our inclusion criteria, we also added the primary trial to the dataset. We identified five such studies, three of which were published between 2010 and 2014. We included six studies published from 2015 to 2020 identified in the PrEP literature (e.g. references of other publications or adjacent studies in a search database) that met our criteria for inclusion. All studies were imported into Endnote (X9) to remove duplicates, book chapters, conference abstracts and theses. The resulting set was imported into Abstrackr  for title and abstract review .Authors  conducted an initial screening of 20 abstracts to arrive at consensus on studies for inclusion and exclusion, followed by screening of all abstracts using Abstrackr Beta . Full‐teAuthors used spreadsheet software to extract the following information: lead author, year of publication, country of study, sample size, study population, study design, study objective, HIV prevention approach, approach category, mental health component in intervention, HIV prevention outcome, mental health assessment used, and key HIV prevention/mental health finding. PC and JV conducted a second‐level review of all eligible manuscripts. Due to the heterogeneity of our sample in terms of intervention type, outcome and populations, we conducted a qualitative synthesis of study findings.33.1A total of 3340 articles were identified through search criteria. After removing duplicates, book chapters, conference abstracts and theses, and restricting to 2015 to 2020 publication years, 1023 articles were eligible for the title/abstract screen. A total of 148 articles were full‐text reviewed and 46 were eligible to be included in this review. We added 11 articles after full‐text review, yielding a total of 57 articles in this review  testing the efficacy of an HIV prevention intervention with or without a mental health component, two were secondary analyses of randomized controlled trials, five were longitudinal studies examining risk reduction behaviour and 17 were cross‐sectional studies assessing the relationship of mental health status and HIV risk or prevention. The studies represent samples from 26 countries: United States (n = 23), Kenya (n = 5), South Africa (n = 4), Brazil (n = 5), Ecuador (n = 1), Peru (n = 3), Thailand (n = 3), Burkina Faso (n = 2), Belgium (n = 1), Canada (n = 2), China (n = 1), Colombia (n = 1), Côte d’Ivoire (n = 1), England (n = 2), Ethiopia (n = 1), India (n = 1), Jamaica (n = 1), Malaysia (n = 1), Mali (n = 1), Mexico (n = 1), Nepal (n = 1), Togo (n = 1), Uganda (n = 1), Vietnam (n = 1), Zambia (n = 1) and Zimbabwe (n = 1). The majority of studies recruited MSM (n = 19) and adolescents (n = 19). The remainder were transgender (n = 9), young women (n = 6), people with SMI (n = 3), FSW (n = 3), PWID (n = 3), mental health facilities (n = 1) and incarcerated persons (n = 1). The studies examined a range of HIV prevention approaches: 22 focused on HIV risk behaviour interventions, 12 on oral PrEP, 10 on condom use, 10 on voluntary counselling and testing, 6 on HIV knowledge/education, and 2 considered structural HIV prevention approaches such as free schooling and conditional cash transfers. Fewer studies examined biomedical interventions like treatment of sexually transmitted infections (STI) (n = 4), systems interventions (n = 1) and VMMC (n = 1). The studies measured mental health outcomes using screening assessments (n = 30), structured clinical interviews (diagnostic assessments) (n = 10) and other self‐report items (n = 32). We present study findings according to three population groups  and service integration, first describing trials and quasi‐experimental studies followed by non‐experimental designs for each group.3.2Nineteen studies enrolled adolescents or young women and reported HIV prevention and mental health‐related factors as explanatory or outcome variables Table . Depress3.2.1Thirteen studies tested the efficacy of an HIV prevention intervention utilizing theory‐based skills building and reported both HIV and mental health outcomes among adolescents and young adults Table . All stuSeveral studies clearly articulated behavioural or mental health symptom targets and integrated dialectical behaviour therapy techniques , 42, emoParental mental health outcomes were indirectly related to adolescent HIV risk behaviours in two behavioural intervention studies , 54, 55.3.2.2One randomized controlled trial tested a cash transfer intervention, which significantly reduced depressive symptoms among young men only and delayed sexual debut among young men and women .3.2.3Five studies assessed mental health as an explanatory variable for HIV risk or preventive behaviours Table . DepressDepressive symptoms were also measured in the context of VMMC service uptake and parental consent among circumcised and uncircumcised adolescents . CircumcA South African study of HIV risk, adverse childhood experiences (ACEs), adolescent mental health, and free schooling for adolescent girls, found that relationships between ACEs and HIV risk behaviour were mediated by internalizing and externalizing symptoms . Free sc3.3Twenty‐six studies enrolled members of key populations and reported HIV prevention outcomes and mental health‐related factors as explanatory or outcome variables Table .3.3.1Eight studies reported the efficacy of a theory‐based intervention for HIV risk reduction , 69, 70.3.3.2Analysis of a PrEP safety trial among MSM and TGW revealed a high prevalence of moderate depression across participants in both study arms, but no difference in depression‐related adverse events or reports of suicide attempts and self‐harm between participants in the PrEP and placebo arms . A sub‐g3.3.3Eighteen studies examined mental health as a correlate of HIV risk reduction in studies of HIV or STI testing and counselling, condom use, and awareness of, adherence to or engagement with PrEP Table .fewer symptoms of a mental disorder were associated with HIV or STI testing, and greater severity of symptoms was associated with less testing [previous diagnosis of depression [Of these, seven examined the relationship of mental health variables to HIV or STI testing in cohorts and community samples Table . In four testing , 75, 76.pression . In contpression . Notablypression .Several studies demonstrated that poorer mental health  was associated with less consistent condom use or less perceived condom self‐efficacy among MSM , young wTwo studies showed that distinct categories of mental disorder symptoms were associated with lower adherence to PrEP: anxiety symptoms and a history of childhood trauma , baselin3.4Three studies enrolled people with SMI in a clinical trial or cross‐sectional surveys.3.4.1A randomized controlled trial tested a sexual health promotion intervention among adults with SMI  Table 33. Partic3.4.2Two Brazilian studies examined people with SMI and HIV risk correlates Table . One stu3.5Two studies in our sample described distinct approaches to HIV prevention and mental health service integration Table . One assEnd the Epidemic activities in the state . Compared to past surveys, fewer mental health programme directors reported integration of HIV services and psychiatric services, and fewer identified themselves as fully integrated in 2017 compared to 2004, despite more programmes reporting larger caseloads of people with HIV or AIDS.A survey of mental health programme directors in New York State showed that a majority of programmes treated people known to have HIV, assessed HIV risk, and provided HIV educational materials, and just over half referred people for HIV testing . Between3.6Thirteen studies in our sample describe HIV prevention interventions  with an embedded or linked mental health component(s) that reduced HIV risk behaviours and produced a more favourable mental health outcome Table . The majTo our knowledge, this is the first review of global research on mental health and HIV prevention among adolescents and young women, key populations and people with SMI that includes multiple prevention modalities. We aimed to understand how mental disorder symptoms influence risk for HIV and preventive intervention outcomes among three populations vulnerable to HIV infection. Overall, this selection of studies, dominated by behavioural interventions to reduce sexual risk, suggests that poorer mental health is associated with HIV risk behaviour. Study findings help to answer two questions: 1) What do we know about the relationship between mental health and HIV prevention behaviours and 2) What do we know about the interventions that address mental health in the context of HIV prevention?3.7Having symptoms of a mental health condition was more often associated with fewer HIV prevention behaviours. In all but one study, depression impaired PrEP adherence. In the majority of HIV testing and counselling studies, having fewer symptoms of a mental disorder increased HIV testing. Depressive, anxiety and trauma symptoms usually reduced the likelihood of condom use or condom self‐efficacy.In a subset of studies, depressive symptoms (current or past) were associated with a greater likelihood of getting an HIV test, and with PrEP initiation . In thesThe relationship between HIV risk and mental health is sometimes indirect. For adolescents, parent–child communication skills, parenting styles and parental mental health status influenced successful sexual communication, which in turn reduced HIV risk . Consist3.8Prevention scientists emphasize the importance of combination prevention and comprehensive, layered approaches that address contextual and individual risk factors for HIV prevention , 97, 98.The thirteen studies in Table Notably, few new intervention studies for people with SMI have been published since 2012, and studies from Africa, Asia, or Latin America are scarce . Althoug3.9The Global AIDS Targets for 2025 call for «people‐centred and context‐specific integrated approaches» so that 90% of people at high risk of HIV are linked to services for mental health, sexual and gender‐based violence and other relevant care . The res3.10Our study has several limitations. The included studies were heterogeneous and precluded the use of a meta‐analysis of the results and effect sizes. Our review was systematized, though not systematic, and we may not have captured all representative studies. We did not rate the quality of the studies or conduct a bias assessment, but reported findings qualitatively. Although we captured studies from a diverse set of countries, the majority of studies are from the United States and interventions reflect the contextual specificities of the study populations and settings. Self‐report of HIV risk behaviours, non‐randomized study designs and small sample sizes may bias some study findings. Despite these weaknesses, the included study results reflect a broad range of countries and support the assertion that poorer mental health is linked to fewer HIV prevention behaviours and activities globally and that integrated interventions can reduce risk.4Consistent with previous studies, current evidence suggests that mental health conditions are more often associated with poor HIV prevention outcomes, and integrated approaches are urgently needed to address overlapping vulnerabilities among key populations, vulnerable groups and individuals with SMI. Our review contributes a new synthesis of global literature on mental health and HIV prevention, spanning a broad range of prevention modalities; studies from high‐, middle‐ and low‐income countries; and diverse samples of key populations, high‐risk groups, and people with SMI. We highlight the components of interventions that address symptoms of mental illness or psychological processes that influence mental health status. Importantly, these findings, in concert with the broader global mental health literature , 115, suThe authors have declared no conflict of interest.PC, TC and JV developed search criteria. TC, JV and LO completed the primary title/abstract review of studies. PC, TC, JV, LO, LC and JS completed the secondary full‐text review of studies. PC, TC, JV, LO, LC, BW and JS assisted with data coding and analysis. PC, TC, JV, LO, LC, JS and CK contributed to development, writing and editing of this manuscript.ACEs, Adverse childhood experiences; ADS, Affect Dysregulation Scale; AE’s, Adverse events; AGYW, Adolescent girls and young women; AI/AN, American Indian/Alaska Native; BPRS, The Expanded Brief Psychiatric Rating Scale; BSI, Brief Symptom Inventory; CASA, The Child and Adolescent Services Assessment; CDI, Children's Depression Inventory; C‐DISC‐IV, Computerized Diagnostic Interview Schedule for Children; CES‐D, Center for Epidemiological Studies Depression Scale; CES‐D‐R, Center for Epidemiological Studies Depression Scale Revised; CMD, Common mental disorders; DANVA, Diagnostic Analysis of Nonverbal Accuracy; DASS, Depression, Anxiety, and Stress scale; DERS, Difficulties in Emotion Regulation Scale; DISC, Diagnostic Interview Schedule for Children 4.0; DSM‐IV, Diagnostic and Statistical Manual of Mental Disorders, fourth edition; DTS, Davidson Trauma Scale; EMR, Electronic medical record; EPDS, The Edinburgh Postnatal Depression Scale; ER, Emotion regulation; FSW, Female sex worker; GAD, Generalized Anxiety Disorder; GHQ12, General Health Questionnaire; GSI, Global Severity Index; HADS, Hospital Anxiety and Depression Scale; HIV, Human Immunodeficiency Virus; HRQoL SF‐12, Health‐Related Quality of Life SF‐12; HSCL, Hopkins Symptom Checklist; HSCL‐D, Hopkins Symptoms Checklist for Depression; HTC, HIV Testing and Counselling; LBQ, Lesbian Bisexual Queer; MADRS, Montgomery‐Asberg Depression Rating Scale; MASC, Multi‐Dimensional Anxiety Scale for Children; MH, Mental health; MINI PLUS, Mini International Neuropsychiatric Interview – PLUS; MINI, Mini International Neuropsychiatric Interview; MINI‐Kid, Mini International Psychiatric Interview for Children and Adolescents; MSM, Men who have sex with men; NR, Not reported; OVC, Orphans and vulnerable children; PHQ, Patient Health Questionnaire; PrEP, Pre‐Exposure Prophylaxis; PROMIS, Patient‐Reported Outcomes Measurement Information System Depression—Short Form 8b; PTSD, Post‐Traumatic Stress Disorder; PWID, Persons who inject drugs; QIDS, Quick Inventory of Depressive Symptomatology; RCMAS, Revised Children's Manifest Anxiety Scale; RCT, Randomized control trial; SCID, Structured Clinical Interview for DSM‐IV Diagnosis; SCL‐90R, The Symptom Checklist‐90 Revised; SDQ, Strengths and Difficulties Questionnaire; SI, Suicidal intent; SITBI 2.0‐SF, Self‐Injurious Thoughts and Behaviour Interview 2.0 ‐ Short Form; SMI, Serious Mental Illness; SSQ, Shona Symptom Questionnaire; STI, Sexually transmitted infections; SW, Sex worker; TAS, Toronto Alexithymia Scale; TasP, Treatment as prevention; TG, Transgender person; TGW, Transgender women; THQ, Trauma History Questionnaire; UNICEF, United Nations Children's Fund; VMMC, Voluntary medical male circumcision; YSR, Youth Self Report.Additional file S1. Search strategy.Click here for additional data file.


Inflammation and the immune system significantly impact the development, progression, and treatment response of hepatocellular carcinoma (HCC). This retrospective study investigated the neutrophil‐to‐lymphocyte ratio (NLR) as a prognostic biomarker in Western patients with HCC in the setting of chronic viral hepatitis.Patients diagnosed with HCC from 2005 to 2016 were selected from a tertiary care institution. NLR was calculated within 30 days prior to treatment and dichotomized at the median. Kaplan–Meier overall survival (OS) curves and Cox hazard proportional models were utilized. Tumor and liver reserve parameters were included in multivariable analyses (MVA).p < 0.0001). Log‐transformed NLR was associated with decreased OS, after multivariable adjustment for confounders . Viral hepatitis was identified as an NLR effect modifier: in nonviral hepatitis (n = 210), low NLR was associated with higher median OS versus high NLR . This was decreased in viral hepatitis (n = 371) . Further, the interaction term between hepatitis and log‐transformed NLR was significant (p = 0.0274) on MVA.A total of 581 patients met inclusion criteria  with median OS = 34.9 mo. 371 patients (63.9%) had viral hepatitis, of which 350 had hepatitis C (94.3%). The low‐NLR group (<median NLR = 2.45) demonstrated higher median OS of 45.6 mo versus the high‐NLR group (median OS 23.9 mo, Lower baseline NLR was associated with increased overall survival in HCC. Viral hepatitis serves as an effect modifier of NLR, attenuating its prognostic relevance in this hepatitis C‐predominant population. Lower baseline NLR was associated with increased overall survival in HCC. Chronic viral hepatitis serves as an effect modifier of NLR, attenuating its prognostic relevance in this hepatitis C‐predominant population. Increased levels of tumor‐infiltrating CD8+ T cells have been associated with improved survival in HCC,Despite increasing interest in the NLR and other immune biomarkers in HCC, few have examined their relevance in a Western, HCV‐predominant population. Therefore, the aim of our study was to investigate the relevance of the NLR as a prognostic biomarker in an HCV‐predominant HCC population, with an emphasis on chronic viral hepatitis.22.1Institutional review board approval was obtained prior to the study, and the protocol was conducted in compliance with the Health Insurance Portability and Accountability Act. We retrospectively analyzed patients from the cancer registry of a single urban academic center diagnosed with HCC based on histopathological or radiological assessment between 2005 and 2016 who received treatments through the hospital system. Informed consent for data usage was obtained from patients at the time of initial treatment. Treatment allocation was determined by a multidisciplinary tumor board and was stratified into systemic therapy, transarterial chemoembolization (TACE), ablation, combined locoregional therapy (combo LRT), resection, and transplantation. Systemic therapy consisted of patients who received chemotherapy with or without radiation. Patients who received both TACE and ablation within a 2‐month period were classified as combo LRT, and patients who received systemic therapy in addition to TACE or ablation were classified as TACE and ablation, respectively. Patients who received resection or transplant following other therapies were classified as resection and transplant, respectively. We excluded individuals under the age of 18, patients with incomplete treatment data, those receiving palliative therapy, and those with a pathologic diagnosis of hepatocholangiocarcinoma.2.2Variables reported in the cancer registry included age, gender, ethnicity, American Joint Committee on Cancer (AJCC) staging, treatment status, and vital status. Further treatment and clinical data were acquired through electronic medical record review. Baseline laboratory values and clinical data were used to calculate Child–Pugh and Model for End‐Stage Liver Disease (MELD) scores and Barcelona Clinic Liver Cancer (BCLC) staging at diagnosis. The Charlson Comorbidity Index (CCI)Baseline NLR (neutrophils/lymphocytes), PLR (platelets/lymphocytes), and SII (neutrophils*platelets/lymphocytes) were calculated using lab values drawn within 30 days prior to treatment. Thrombocytopenia was defined as platelets <150,000/μl.2.32 test and continuous variables using the Wilcoxon rank sum test. Survival was estimated using the Kaplan–Meier method and log‐rank test, as well as the Wilcoxon test where survival curves crossed. The Cox proportional hazards method was used to identify predictors of overall survival (OS). Significant predictors in univariate Cox models were tested in multivariable models (MVA) to address confounding. Non‐normally distributed continuous variables were log‐transformed for survival modeling.Pretreatment immune biomarkers were dichotomized at the median for visualization. For other survival analyses, biomarkers were log‐transformed and evaluated as linear predictors. Categorical variables were compared using the χp‐values reported are two‐sided. Calculations were performed and figures created using JMP Pro, R, and GraphPad Prism.An alpha‐level of <0.05 was considered statistically significant. All 33.1n = 333, 57.3%), HBV , combined HBV/HCV , alcohol , NASH , and other . The majority of patients had Child–Pugh A disease . TACE was the most frequent treatment utilized . Seventy patients received systemic therapy (12.0%), of whom 63 (90%) received sorafenib. Additional baseline patient and tumor characteristics are provided in Table A total of 581 patients met inclusion criteria   included older age, increased comorbidities, nonviral hepatitis, higher Child–Pugh score, advanced BCLC stage, advanced AJCC stage, increased tumor size, multifocal tumors, bilobar tumor burden, vascular invasion, and extrahepatic metastases as well as systemic therapy and increased alpha‐fetoprotein (AFP). On multivariable analysis, decreased survival was associated with Child–Pugh B disease, BCLC stage D, AJCC stage IV, bilobar tumors, nonviral hepatitis, systemic therapy, and AFP (p < 0.05). , ablation (36.5 mo), combo LRT (27.9 mo), TACE (22.2 mo), and systemic therapy  for the cohort was 3.2*10^3/μl, with median absolute lymphocyte count (ALC) of 1.3*10^3/μl and median platelet count of 117.5*10^3/μl. The median NLR was 2.45, median PLR was 95.28, and median SII was 290.05. As depicted in Figure p = 0.0048). Decreased NLR was associated with lower comorbidity indices, higher rates of viral hepatitis; lower MELD and Child–Pugh scores; lower BCLC and AJCC staging, lower tumor size, less vascular invasion, higher rates of unilobar tumors, and fewer extrahepatic metastases (p < 0.05). The low‐NLR cohort had lower rates of alcoholic hepatitis (7.6%) or NASH (7.2%) versus patients in the high‐NLR group  and also had higher rates of ablation, combo LRT, and resection and lower rates of systemic therapy, TACE, or transplant as compared to the high‐NLR group.Patients in the low‐NLR group had higher rates of African American race (16.9%) or Hispanic ethnicity (17.2%) than patients in the high‐NLR group . The low‐NLR group (<median NLR = 2.455) demonstrated higher median OS of 45.6 mo versus the high‐NLR group ; TACE ; and resection . While a similar trend was seen in the low‐NLR group for ablation, combo LRT, and transplant, these did not reach statistical significance.Log‐transformed NLR was associated with decreased OS after multivariable adjustment for confounders , of which 333 (89.8%) were HCV, 21 (5.6%) were HBV, and 17 (4.6%) were HBV/HCV coinfection. A total of 137 HCV patients (39.3%) received direct‐acting antiviral therapy (DAA), with 31 (22.3%) having started DAA therapy prior to HCC diagnosis. The viral hepatitis group had higher survival from diagnosis of HCC (median OS 38.9 mo) versus nonviral hepatitis patients .Sixty‐four percent of patients had viral hepatitis (p < 0.05) and had lower comorbidity indices, lower AJCC staging, higher median AFP, and decreased median tumor size (p < 0.05) (Table p < 0.01). While patients with viral hepatitis had higher rates of BCLC stage 0 or A disease and lower rates of BCLC stage B disease compared to patients with nonviral hepatitis, rates of BCLC stage C and D disease were similar between the two groups. Patients with and without viral hepatitis had similar distributions of Child–Pugh score, MELD score, unilobar versus bilobar tumor burden, multifocal tumors, vascular invasion, and extrahepatic metastases.Patients with viral hepatitis were more likely to be younger, male, and African American (5) Table . Patientp < 0.0001) and higher platelet counts . Median ALC was similar between the two groups . Patients with viral hepatitis had higher rates of thrombocytopenia (68.5%) versus nonviral hepatitis . The median NLR was higher in nonviral hepatitis patients (3.0) versus viral hepatitis patients , as was the median PLR  and the median SII .HCC patients with viral hepatitis had lower ANC versus patients with nonviral hepatitis . In nonviral hepatitis patients, low NLR was associated with increased survival . However, in the presence of viral hepatitis, this prognostic relevance was attenuated . Similar results were found for the HCV subgroup; however, the interaction term between HCV status and the log‐transformed NLR was nonsignificant (p = 0.0537), likely due to lower power in the HCV subgroup due to smaller sample size.On multivariable analyses, viral hepatitis was identified as an effect modifier for NLR: the interaction term between hepatitis status and log‐transformed NLR was significant on MVA , as well as higher rates of thrombocytopenia. Liver disease burden as assessed by Child–Pugh and MELD scores was similar between the two groups, suggesting that the increased thrombocytopenia of the viral hepatitis cohort may be related to factors beyond cirrhosis.While decreased NLR in our study was associated with fewer comorbidities (possibly due to decreased systemic disease burden and decreased systemic inflammation), less advanced liver disease, less advanced cancer staging, and smaller tumors, log‐transformed NLR remained an independent predictor of overall survival on multivariable analysis. This suggests that the mechanism of action of the NLR may not be through severity of liver disease or stage of HCC but through other mechanisms.4.3The interplay between cancer formation and the immune system is complex. For example, neutrophils have been found to have both pro‐tumor and antitumor properties.Although our study focused solely on circulating markers of inflammation, it is plausible that the NLR may be associated with changes in the tumor microenvironment. There is no current consensus about how the NLR affects the immunologic composition of HCC. A meta‐analysis by Zhang and colleagues reported that both increased peripheral blood T‐regs and increased intratumoral T‐regs were associated with poorer survival in HCC.Of the treatments examined in subgroup analyses, the NLR had the strongest effect in systemic chemotherapy patients, with survival in the low‐NLR group being almost three times as high as survival in the high‐NLR group. The majority of these patients (90%) received sorafenib, which is a tyrosine kinase inhibitor (TKI) that inhibits tumor angiogenesis and proliferation while increasing apoptosisFuture studies examining the NLR could focus not only on sorafenib, but also on the several other oral systemic therapies which have been recently approved. In addition, when studying the efficacies of systemic therapies, it may be prudent to stratify analyses by viral or nonviral HCC, particularly since subgroup analyses of the SHARP trial demonstrated significant benefit in HCV patients,4.4Our study included a diverse, well‐characterized cohort regarding age, gender, race, and ethnicity as well as diverse treatment modalities. Our sample size is a limiting factor in subgroup analyses; however, our HCC cohort is among the largest studies in which NLR has been examined. While this study relied on archived records, we limited the NLR values to within 30 days of HCC treatment in order to improve standardization. Our study had low rates of DAA initiation or completion prior to HCC diagnosis. HCV‐predominant HCC cohorts with higher rates of DAA completion prior to HCC diagnosis may demonstrate varying impact of the NLR. This study was conducted among a U.S. population, and results may differ in other parts of the world.5Lower baseline neutrophil‐to‐lymphocyte ratios are associated with decreased survival in Western patients with HCC. The neutrophil‐to‐lymphocyte ratio is a superior prognostic marker to the platelet‐to‐lymphocyte ratio and systemic immune‐inflammatory index. The effect of the NLR is decreased in patients with a history of viral hepatitis in this cohort of hepatitis C‐predominant HCC. Further studies are warranted to investigate the impact of chronic viral hepatitis on tumor inflammation, immune response, and survival in HCC, particularly at a local or tumor level.The author has no conflicts of interest to report for this article. The author certifies that they have no affiliations with or involvement in any organization or entity with any financial interest in the subject matter or materials discussed in this manuscript. HSK has served on Advisory Boards for Boston Scientific, Sirtex, Genentech, Eisai, Amgen, and Bayer, and was supported of research device support from Galil and database from Flatiron Health. JU received speaker fees from Bayer Vital GmbH on a subject not related to the study at hand.CMS, JU, JML, and HSK contributed to conceptualization, formal analysis, methodology, and reviewing and editing the manuscript. CMS was responsible for data curation, funding acquisition, and writing the draft manuscript. JKL, JP, TT, and SS contributed to reviewing and editing the manuscript. HSK supervised this project. All authors read and approved the final manuscript.Fig S1Click here for additional data file.Fig S2Click here for additional data file.