Background: Hyzetimibe is a novel inhibitor of cholesterol that specifically targets the NPC1L1 gene. Significant inter-individual variability suggests the existence of an abundance of poor responders and non-responders. In addition, the current literature is inconsistent and controversial regarding the potential impact of the Niemann-Pick C1-Like 1 (NPC1L1) gene on low-density lipoprotein cholesterol (LDL-C) reduction. In light of these concerns, we performed a high-quality clinical trial to investigate the specific characteristics of NPC1L1 gene variation on LDL-C reduction.Methods: This was a multicenter, randomized, double-blind, placebo-controlled, clinical trial with a factorial design. Qualified patients were randomly assigned to one of six treatments: placebo, hyzetimibe (10 or 20 mg), atorvastatin, and atorvastatin plus hyzetimibe (10 or 20 mg). Fasting blood samples were collected and genotyped, and the concentrations of LDL-C and the targeted drug trough were determined to investigate the association between the NPC1L1 gene expression and the reduction of LDL-C.Results: In total, 727 individuals were initially recruited; of these, 444 were eligible to begin the trial. We identified one SNP (g1679C > G) that exerted significantly different impacts on LDL-C levels. As monotherapy, CC carriers experienced significantly higher reductions in the mean LDL-C (−23.99%) than either the GG  or GC  carriers in the hyzetimibe (20 mg) group. In contrast, when co-administered with atorvastatin, GC carriers experienced greater LDL-C reduction than non-GC carriers (-52.23% vs. −45.03%) in the hyzetimibe (20 mg) plus atorvastatin group. Furthermore, the proportions of individuals experiencing a reduction in LDL-C by >50% increased as the dose of hyzetimibe increased from 16.1% to 65.4%.Conclusion: The g1679C > G SNP in the NPC1L1 gene is critical and exerts a differential impact on the response to hyzetimibe treatment. Heterozygotic patients respond with poor efficacy when treated by monotherapy but show good responses in terms of LDL-C reduction when hyzetimibe was co-administered with atorvastatin. To treat hypercholesterolemia in a precise manner with hyzetimibe, it is necessary to identify genotype patients for the g1679C > G SNP. We also highlight the potential necessity for identifying the appropriate subjects to be treated with ezetimibe.https://clinicaltrials.gov/], identifier [CTR20150351]Clinical Trial Registration: [ The treatment of dyslipidemia has become critical for the early prevention and treatment of cardiovascular diseases, and the reduction of low-density lipoprotein cholesterol (LDL-C) plays a critical role in reducing the risk of cardiovascular events in both primary and secondary prevention .14C-radiolabeled hyzetimibe revealed that a change in the hydroxyl group within the drug structure made it easier for hyzetimibe to combine with glucuronic acid, thus leading to the increased urinary excretion of hyzetimibe than ezetimibe (NPC1L1)  is the second inhibitor of cholesterol absorption to be developed in China and was approved for marketing by the National Medical Products Administration of China (NMPA) in June 2021. A pharmacokinetic study of oral zetimibe . Hyzetim(NPC1L1) , which p(NPC1L1) . Data obsorption . Therefo. Ezetimibe monotherapy has shown to reduce the levels of LDL-C by 15–20%; an additional reduction of −13% to −20% could be attained when ezetimibe was combined with statin therapy, depending on the concentration of statin administered (Ezetimibe is another inhibitor of cholesterol absorption and was first approved for marketing by the Food and Drug Administration (FDA) in 2002 . Ezetiministered .NPC1L1 gene on LDL-C reduction in response to ezetimibe (versus atorvastatin (VYVA) clinical trial. The EASE study was designed to evaluate the effects of ezetimibe, in combination with a stable regimen of statin therapy . In contrast, the VYVA study aimed to investigate the effects of a combination of ezetimibe and simvastatin in comparison with atorvastatin in patients with hypercholesterolemia.Pharmacogenomic studies were carried out in over 1,000 patients with hypercholesterolemia to investigate the impact of polymorphisms in the zetimibe . HoweverNPC1L1 gene. Furthermore, neither of these studies considered ezetimibe monotherapy; this made it difficult to investigate the specific response to ezetimibe monotherapy. In addition, in the EASE cohort, ezetimibe was combined with statins (any dose and any brand); differential negative feedback mechanisms arising from different statins and doses, along with the complex background of statin therapy, made it difficult to determine the precise effects of these treatments.These trials involved only a single dose of ezetimibe; this made it difficult to identify the specific association between the drug dose and the response exerted by the genetic variation in the n = 65, p < 0.07) in Caucasian familial hypercholesterolemia and by −29.2 ± 1.0% and −33.7 ± 2.6%  in patients with primary hypercholesterolemia with the CC genotype and non-CC genotype of the g1679C > G SNP, respectively.NPC1L1 mutations had a 53% relative risk reduction for coronary heart disease and had lower levels of LDL-C than non-carriers (p = 0.04).Subsequently, the NPC1L1 gene and proteome in an exceptional responder to ezetimibe and attempted to identify genetic variations that caused functional changes. The combination of L52P-NPC1L1 and I300T-NPC1L1 amino acid changes caused a functional reduction in cholesterol uptake in the presence of ezetimibe, which was considered as a key aspect of the exceptional response.NPC1L1 on the effect of cholesterol absorption inhibitors; existing data are inconsistent and unclear. Consequently, there is a critical need to carry out a high-quality clinical trial to investigate the characteristics of gene variation with exceptional responders or poor responders to drug treatment. Thus, we carried out a multicenter, randomized, double-blind, balanced-parallel, and placebo-controlled trial in 727 Chinese patients with primary hypercholesterolemia. For each patient, we characterized variability in the DNA sequence of the NPC1L1 gene and subsequently investigated the association between NPC1L1 and LDL-C reduction induced by hyzetimibe treatment.Clearly, there is a lack of specific data relating to the potential impact of gene variations in The trial was carried out between 2015 and 2019, and patients with primary hypercholesterolemia from 43 medical institutions across China were enrolled. The inclusion criteria were as follows: individuals aged 18–70 years with primary hypercholesterolemia, defined as a calculated LDL-C of 3.36–4.88 mmol/L, following dietary control for >4 weeks; individuals in which the difference between two LDL-C concentrations, measured more than 1 week apart during the washout period, was less than 12%; and individuals who had not received any cholesterol-lowering drugs, such as statins, for 6 weeks prior to the trial. All patients provided written and informed consent before enrollment.The exclusion criteria were as follows: homozygous familial hypercholesterolemia; severe heart, liver, lung, kidney, and other important organ diseases; serious muscle abnormalities and neuropathies; uncontrolled endocrine disease known to influence serum lipids or lipoproteins; ezetimibe intolerance; patients who were diabetic and aged >40 years; diabetes with cardiovascular risks; blood pressure ≥160/100 mmHg; thyroid dysfunction; a history of malignant tumors; triglyceride ≥3.99 mmol/L (350 mg/dl); known impairment of renal function; active or chronic hepatic or hepatobiliary disease; atherosclerotic cardiovascular disease; uncontrolled cardiac arrhythmia; and HCV or HBsAg positive. We also excluded patients who were taking fibric chemicals such as fenofibrate, gemfibrozil, probucol, warfarin, corticosteroids, cyclosporine, or other immunosuppressives within 12 weeks prior to the trial.The entire trial was conducted in accordance with the principles of the Declaration of Helsinki for biomedical research involving human subjects and according to the International Conference on Harmonization Guidelines for Good Clinical Practice.A multicenter, randomized, double-blind, balanced-parallel, and placebo-controlled group trial was designed carefully and carried out in a strict manner. The protocol was reviewed and approved by the Independent Ethics Committee of the Second Affiliated Hospital Zhejiang University School of Medicine and other participating medical institutions. The clinical trial was approved by the National Medical Products Administration of China . The clinical trial was registered on the Chinese Clinical Trial Registration and Information Platform (Registration number: CTR20150351).On the first day of informed consent, patients received dietary counseling and were randomly assigned by a centrally managed service (the Interactive Web Response System) to receive drug treatment according to randomized stratification by site, age, sex, and baseline LDL-C level. The 4-week screening phase included the washout of previous lipid-modifying drug therapy and was instructed to follow a stricter diet throughout the trial. At week 0, patients were randomly assigned to one of six treatments for 12 weeks: placebo (PLB), hyzetimibe 10 mg (HS25-10 mg), hyzetimibe 20 mg (HS25-20 mg), atorvastatin 10 mg (ATO), atorvastatin (10 mg) plus hyzetimibe 10 mg (ATO + HS25-10 mg), and atorvastatin (10 mg) plus hyzetimibe 20 mg (ATO + HS25-20 mg).Fasting blood samples were collected at the baseline and weeks 2, 4, 8, and 12. LDL-C levels were measured by using an AU5800 automatic Beckman biochemical analyzer (Beckman Coulter) at the central laboratory.Plasma concentrations of hyzetimibe, hyzetimibe-M1 , along with atorvastatin trough concentrations, were determined by liquid chromatography (Shimadzu) and mass spectrometry (AB SCIEX) at the central laboratory.DNA samples were sequenced by next-generation sequencing performed by BGI . The coding genes for the target NPC1L1 protein were sequenced, including the exons, intron junctions, 3′-untranslated region (3′- UTR), 5′-UTR, and the promoter.versus hyzetimibe  or atorvastatin). Statistical analyses were performed by SAS software . A p value less than or equal to 0.05 was considered statistically significant.The percentage reduction of the LDL-C level from the baseline to final assessment was considered as the primary efficacy end point and was analyzed by a two-way analysis of variance (ANOVA) model that extracted effects due to placebo and treatment (hyzetimibe 10 and 20 mg). Next, we performed a series of data comparisons ) NPC1L1; patients were excluded due to incomplete data relating to C trough and LDL-C, non-target substances in the plasma, or the absence of genotyping data ; the MAFs for these SNPs all exceeded 10% in Chinese patients with primary hypercholesterolemia. However, only those with the g1679C > G (rs2072183) genotype were associated with the lipid-lowering effect of hyzetimibe. The frequency of the g1679C > G genotype was similar to that in a previous Japanese population (p > 0.05) but was statistically different from the frequency recorded in a healthy population of Chinese and Japanese patients (p < 0.001), as well as in Canadian patients with primary hypercholesterolemia (p < 0.001). These findings indicated that there was a significant difference in the frequency of the g1679C > G variant when compared between healthy Chinese subjects and those with dyslipidemia; there was a clear genetic variation between Chinese and Canadian patients with dyslipidemia (ipidemia  (Simon eipidemia .p < 0.01). The reduction of LDL cholesterol by hyzetimibe occurred early (2 weeks) and was maintained throughout the 12-week treatment period. The coadministration of hyzetimibe and atorvastatin resulted in a significantly greater mean reduction of LDL-C than either atorvastatin alone or hyzetimibe alone (Treatment with hyzetimibe (10 and 20 mg) resulted in a mean percentage reduction from the baseline to end point in the plasma concentration of LDL cholesterol  as compared to a decrease of only 3.12% in the placebo group  in the hyzetimibe (20 mg) group showed similar reductions in the levels of LDL-C reduction at 0.77 mmol/L  in the hyzetimibe (20 mg) plus atorvastatin group (p = 0.051) with respect to the reduction of LDL-C; patients exhibiting the GC genotype exhibited a greater reduction in LDL-C than those with the GG genotype  exhibited a greater reduction in the median level of LDL-C than the non-GC genotypes  than those with the CC (10.71%) or GG (6.48%) genotypes when compared to atorvastatin alone . Similar < 0.001 .NPC1L1 gene and the reduction of LDL-C levels in response to drug treatment with an inhibitor of cholesterol absorption. Our investigation revealed that genetic variations in NPC1L1 exert notable impacts on the extent of LDL-C reduction in response to hyzetimibe treatment in patients with primary hypercholesterolemia.This is the first randomized clinical trial  to demonstrate an association between the NPC1L1 gene and is embedded within the coding region. This SNP was in linkage disequilibrium with known functional variants, such as NPC1L1 -762T > C, an important transcription factor binding site, and −18C > A, in close proximity to a sterol regulatory element, thus suggesting that this SNP is related to the transcriptional level of NPC1L1; these changes lead to changes in the functionality of the NPC1L1 protein, thus leading to changes in the absorption of cholesterol  or the GG genotype  in patients who were treated with hyzetimibe (20 mg/day). When the effects of the placebo were removed, the reduction in LDL-C levels exhibited a rank order of CC (−20.49%) < GG (14.67%) < GC (−8.46%). The absolute reduction was 0.52 mmol/L for the GC genotype; this was compared to 0.65 mmol/L for the GG genotype and 0.96 mmol/L for the CC genotype.When treated by monotherapy, patients with the CC genotype of the g1679C > G SNP tended to show significantly greater reductions in mean LDL-C (−23.99%) than either the GC genotype  should not be treated by hyzetimibe monotherapy due to poor efficacy. Patients with a homozygous genotype (the CC and GG genotypes) exhibited reductions of LDL-C [0.77 mmol/L (−19.34%)] similar to those of ezetimibe; in particular, the CC genotype exhibited significantly greater reductions in LDL-C than ezetimibe, thus indicating improved clinical benefits.versus −45.03%) in the hyzetimibe (20 mg) plus atorvastatin group  did not show a favorable dose response with regards to a further reduction of LDL-C (a 7.94% increase for CC and a 6.33% increase for GG); this was compared to a 15.29% increase for GC. Patients carrying the GC genotype showed a greater median reduction in LDL-C than those with the non-GC genotype  need to control their cholesterol levels efficiently if they are to reduce the risk of cardiovascular events, thus demanding an appropriate lipid-lowering regimen from the beginning of treatment but not to modify according to the efficacy obtained after at least one month.With regards to combination therapy, a previous drug–drug interaction study of atorvastatin and hyzetimibe showed there was no interaction between these drugs; this was also the case for the GC genotype. Thus, when the dose of atorvastatin or hyzetimibe increases, we should see a greater reduction in LDL-C.NPC1L1 gene had differential impacts on the efficacy of cholesterol absorption inhibitors; researchers have been attempting to address unravel these effects for the past 15 years. This is the first study to identify common SNPs and genotypes of the NPC1L1 gene with respect to LDL-C reduction by more than 6%. The inconsistencies of previous studies relating to genotype analysis can be explained by differences in genotype frequency distribution between Eastern and Western ethnic groups and differences in the extent of genotype feedback regulation when different drugs are combined. These results are expected to provide significant guidance for the development of new inhibitors of cholesterol absorption with better efficacy, the design of corresponding clinical protocols, and the optimization of clinical hyperlipidemia treatments.By deploying an appropriate study design, our study found that variations in the NPC1L1 gene exerts significant impact on LDL-C reduction during hyzetimibe treatment. This is because the chemical structure of hyzetimibe is different from that of ezetimibe. Furthermore, the affinities of these drugs to the NPC1L1 protein also differ; the precise impact of the NPC1L1 gene on the action of ezetimibe is yet to be investigated. Second, subjects with the CC genotype achieved levels of LDL-C that were two-fold lower in response to hyzetimibe monotherapy treatment than subjects with the GC genotype (−23.99% versus −13.02%). The extent of LDL-C reduction showed a linear relationship with the dose of hyzetimibe. However, these data were obtained from a small number of patients; future research should involve a larger sample size if we are to fully validate our findings. Third, the frequency of the GG, GC, and CC genotypes was 41%, 46%, and 13% in Chinese patients with hypercholesterolemia; this compares to 5%, 40%, and 55% in Canadian patients with hypercholesterolemia (p < 0.001), respectively. The prevalence of the G allele variants indicates that the g1679C > G SNP exhibits ethnic specificity. However, the effects of hyzetimibe treatment on Caucasian patients with hypercholesterolemia are yet to be fully elucidated. Finally, we identified the notable impact of the 1735 C > G SNP on serum LDL-C levels in response to hyzetimibe treatment; however, we still need to identify the effects of a greater dose of hyzetimibe monotherapy in patients with the CC genotype. In addition, it would be very interesting to investigate whether higher doses of hyzetimibe or atorvastatin (in combination with each other) could achieve a better cholesterol-lowering target.This study has several important limitations that need to be considered. First, the g1679C > G SNP in the versus 1.82], thus suggesting that active preventive strategies aimed at reducing the burden of myocardial infarction and atherosclerotic cardiovascular disease in the growing population aged 70–100 years should be adopted and may need more appropriate drugs to reduce the increased levels of LDL-C. Interestingly, at the same time and in the same journal, Gencer et al. also reported outcomes related to the efficacy and safety of lowering LDL cholesterol in older patients aged ≥75 years. This analysis involved 21,492 individuals from 29 trials; 54.7% were treated with statins, 28.9% were treated with ezetimibe, and 16.4% were treated with PCSK9 inhibitors. The risk of major vascular events in older patients was reduced by 26% per 1 mmol/L reduction in LDL-C; there was no significant difference in this respect when compared to patients aged <75 years. Furthermore, there were no significant differences between statin and non-statin treatments  in The Lancet (2020) . Individeatments . The clieatments . Howevereatments . In addieatments .Therefore, ezetimibe or hyzetimibe monotherapy may be potentially useful for the treatment of older patients with dyslipidemia, especially those with statin-related adverse events  and in Chinese patients with hypercholesterolemia who are intolerant to high doses of statins.The 2018 AHA/ACC treatment guidelines for LDL-C reduction recommended individualized treatment approaches for LDL-C reduction based on an individual patient’s CV risk category . CholestNPC1L1 gene is critical and can exert notably different impacts on the response to hyzetimibe treatment. Patients who are heterozygous (the GC genotype) will exhibit poor efficacy with hyzetimibe monotherapy. However, when co-administered with atorvastatin, patients with heterozygous genotypes achieved much better improvements in LDL-C levels. The results of our trial indicate that the precise treatment of hypercholesterolemia with hyzetimibe would be beneficial for patients and highlights the potential necessity for identifying the most appropriate subjects for treatment with ezetimibe.Elevated cholesterol is an established risk factor for cardiovascular (CV) disease. Hyzetimibe represents a new inhibitor of cholesterol absorption and provides an additional therapeutic option for high-risk patients who cannot meet clinical goals with standard forms of therapy . The IMPROVE-IT trial first demonstrated that the coadministration of an inhibitor of cholesterol and a statin was associated with a lower incidence of cardiovascular events than statin monotherapy . The EWT


Background: Despite the same surgical approach, up to 40% of patients develop chronic postsurgical pain (CPSP) following cardiac surgery, whereas the rest are chronic pain free. This variability suggests that CPSP is controlled partially through genetics, but the genes for CPSP are largely unknown.Aims: The aim of this study was to identify potential CPSP phenotypes by comparing patients who developed CPSP following cardiac surgery vs. those who did not.Methods: A research ethics board–approved, cross-sectional study of post–cardiac surgery pain was conducted at Toronto General Hospital from 2011 to 2015. Patients were recruited to complete a short survey of chronic pain scores and the Short-Form McGill Pain Questionnaire–2. A subset of patients completed a longer survey of eight validated pain phenotyping questionnaires and/or four psychophysical assessments. All surveys and psychophysical testing were conducted after surgery. Patients were stratified by presence of chronic pain and groups were compared using descriptive statistics.Results: Six hundred forty-three patients completed the short form survey. The mean postsurgery assessment time was 41.5 (SD = ±25.1) months. Over a quarter (27.8%) reported CPSP at the chest as a consequence of their surgery. Of patients reporting CPSP, 46.6% reported mild pain (0–3), 35.8% reported moderate pain (4–7), and 17.6% reported severe pain (7–10) in accordance with the numerical rating scale. Patients with moderate and/or severe CPSP were younger, had a greater body mass index, and had higher anxiety sensitivity, pain catastrophizing, and somatization scores.Conclusions: Chronic pain levels after cardiac surgery are associated with anxiety, catastrophizing, and sensory abnormalities in body parts outside the field innervated by injured nerves, indicating the presence of widespread central sensitization to incoming sensory inputs from intact nerves. These dimensions are discrete entities that can be phenotyped with psychometrically sound instruments.3Chronic pain has been coined the silent epidemic of our times, affecting as many as 29% of adults.7Recently, there has been hope that a pain genetics approach may identify new pharmacogenetic targets allowing for development of novel pre-emptive analgesics. This would allow for individualized preventive and palliative treatments that could reduce the number of individuals transitioning to chronicity and alleviate pain in those who have already developed it.The development of chronic postsurgical pain (CPSP) as a consequence of a midline sternotomy following cardiac surgery is a valuable model for genome-wide association studies for chronic pain. The time of the operation is precisely known, and the extent of nerve injury is fairly consistent given the same surgical approach . Moreover, the surgical approach and postoperative analgesic care are standardized within institutions. In addition, CPSP following cardiac surgery presents with distinct neuropathic pain traits that are easily differentiated from preoperative angina pectoris pain that might linger postoperatively.8 Patients undergoing coronary artery bypass graft surgery (CABGS) generally report a significant decline in poststernotomy pain up to 6 months postoperatively. After this period, the pain intensity stabilizes at a level where it remains for years thereafter.10 In one of the largest prospective studies of 1400 patients with chronic post-CABGS pain, Choinière et al. found the incidence of chronic pain post-CABGS to be 40% at 3 months, 22% at 6 months and 17% at 12 months.8 This study identified numerous risk factors for pain post-CABGS, including female gender, younger age, preoperative anxiety, pain catastrophizing and angina, intra-operative opiate use, intense postoperative pain, greater postoperative complications, and worst pain intensity during hospitalization.8Prospective studies of chronic pain after cardiac surgery indicate a 1-year incidence ranging from 11% to 40%.In the present study, we sought to delineate phenotypic differences among patients who developed CPSP following cardiac surgery vs. those who did not using (1) validated pain and psychological questionnaires and (2) psychophysical tests. The results are expected to identify robust intermediate phenotypes for genetic analysis to identify candidate genes as targets of novel preventative and palliative care of chronic postsurgical pain.A cross-sectional study of post–cardiac surgery pain was approved by the research ethics boards at the Toronto General Hospital (TGH) and York University (Research Ethics Board: 10-0852-AE). This study was conducted at TGH from 2011 to 2015. Patients who (1) previously had cardiac surgery at TGH and (2) gave consent for future contact with regard to their postsurgical recovery and novel studies were eligible to participate. Those who consented and gave a previous blood sample to our Genetic Biobank were contacted by telephone by a member of our team at least 3 months after their cardiac surgery. This cross-sectional survey of pain and psychological phenotypes included three separate pain assessments: a short survey, a long survey, and a psychophysical assessment. The goals and study procedures were explained as part of informed consent. Patients had the option to participate in solely the short survey, both short and long surveys, or all three assessments. Patients also consented to use of their blood samples in a future genomic study.Prior to surgery, all patients received standardized peri-operative care. Electronic health records were used to collect patient details, including surgical technique , intra-operative anesthetic variables , cardiovascular intensive care unit postoperative documentation , and hospital discharge date. Participants were phenotyped for chronic postsurgical pain and some additionally completed a series of psychophysical tests. All surveys (short and long) and psychophysical testing were conducted after surgery.11 The McGill Pain Questionnaire–Short Form–2 (SF-MPQ-2) also assessed the quality and intensity of specific pain descriptors.12 The short survey included details to distinguish differences between CPSP, pain that existed pre-surgery and spontaneous pain. In cases where the postsurgical pain was episodic, we collected data on the intensity, unpleasantness, duration, and frequency of a typical pain episode.For patients participating in the short survey alone, verbal consent was obtained and the consent form was sent by mail with a stamped return envelope. Once the consent form was returned, the patient was sent the survey questionnaires and a stamped return envelope. Participants were free to fill out the questionnaires independently or with the assistance of an interviewer via telephone. The short survey utilized numeric rating scales (NRSs) to determine postsurgical pain levels. NRSs provide patients with chronic pain sufficient discriminative power to describe typical pain episode intensity ranging from 0 to 10: mild (0–3), moderate (4–7), or severe (8–10).For patients participating in the long survey, consent was obtained and data were collected in a manner identical to that for the short survey. The long survey consisted of eight validated questionnaires to assess psychosocial factors relevant to the development of chronic postsurgical pain. These included the Anxiety Sensitivity Index (ASI-3), the Pain Catastrophizing Scale (PCS), the Pain Anxiety Symptoms Scale–20 (PASS-20), the Posttraumatic Stress Disorder Checklist–Civilian Version (PCL-C), the Hospital Anxiety and Depression Scale (HADS), the Symptom Checklist (SCL)-90-R–Somatization, Mindful Attention Awareness Scale (MAAS), and the Sensitivity to Pain Traumatization Scale (SPTS).The laboratory invited patients who consented to undergo psychophysical assessment to the laboratory at TGH. The series of tests performed included the following.2) at which the patient first reported pain (using an NRS0–10). PPTs were obtained 2.5 cm away from the surgical scars on the mid-sternum, at the saphenous vein graft, and at control sites on the right and left forearms in the anterior aspect midway between the wrist and elbow. The pressure algometer has been used to obtain PPTs in previous studies.13Pain thresholds in response to mechanical pressure applied to the skin were obtained using a pressure algometer  that registers the applied force. Pressure pain threshold (PPT) was determined as the force . A thermode (16 mm by 16 mm) was applied to the skin on the ventral aspect of the subject’s right forearm and 2.5 cm to the right of the surgical scar on the mid-sternum. When activated, the temperature of the thermode rose or decreased from a baseline temperature of 32°C at a rate of 1°C per second; the patients first detected the temperature as warm or cool, hot or cold, or painfully hot or painfully cold. These sensory detections were made by the patient by depressing a mouse button, which returned the thermode to the baseline temperature at a rate of 1°C per second. Heat and cold pain detection thresholds and thermal detection thresholds to warm and cool stimuli were obtained using the method of limits.15 Participants rested supine on a couch and relaxed with eyes closed and were asked to take the deepest possible inspiration and then breathe normally. Ratings of ongoing spontaneous pain were taken at 1 min after expiration. Two baseline blood pressure  and pulse rate measurements were taken just before the gasp test and at 1 min thereafter, just after the patient reported his or her pain ratings.This test is known to strongly activate the sympathetic system, which may aggravate neuropathic pain in some patients with chronic pain suffering from sympathetically maintained pain.16 This threshold was recorded by the experimenter with a stopwatch as the time from immersion to withdrawal, with a cutoff at 3 min. Participants then used the NRS0–10 to rate the pain intensity and unpleasantness of each stimulus. Changes in cardiovascular nociceptive reflexes caused by the cold pressor pain test were recorded as well and compared to the respective baseline values recorded prior to the cold pressor pain test.Participants immersed the right arm just above the elbow in a coldwater/ice bath (1°C) until they could not tolerate the pain any longer and withdrew at their pain tolerance threshold.Survey data were entered from collected surveys into a Microsoft Excel database and analysis was conducted using IBM SPSS Statistics for Windows, version 25 . Patients were stratified by their chronic pain status  and analyzed using descriptive and inferential statistics. For the short survey, long survey, and psychophysical assessments, univariate general linear models were used to identify variables associated with CPSP status. As a second step, three multivariable multinomial regression models using a backward selection procedure were used to identify factors associated with CPSP status; models were run separately for the short survey, long survey, and psychophysiological data. Age and sex were always forced into the regression models. For the short survey data, variables entered in the multivariable model included body mass index (BMI), preoperative chronic pain status, time from surgery to study consent, and total score on the SF-MPQ-2. For the long survey data, variables entered included BMI, preoperative chronic pain status, and total scores on measures of anxiety sensitivity (ASI-3), anxiety and depression (HADS-D and HADS-A), pain anxiety (PASS-20), sensitivity to pain traumatization (SPTS), posttraumatic stress symptoms (PCL-C), pain catastrophizing (PCS), mindful awareness (MAAS), and somatization (SCL-90-R). As for the psychophysiological data, variables entered included pain scores on the different tests for which sufficient data were collected in each CPSP status category. These variables included mechanical pressure pain threshold–mid-sternal scar, cold pressure pain test 30 s postwithdrawal pain magnitude and unpleasantness ratings, and thermal pain thresholds. Alpha was set at 0.05.In total, 2430 patients were called; 1527 could not be contacted or did not return our call, 185 declined, and 84 did not speak English and were excluded. A total of 634 patients consented to participate and completed the short survey, which included pain scores and the SF-MPQ-2. Of these patients, 367 at least partially completed the long survey and 202 patients completed a set of four psychophysical assessments.N = 367; 57.4%) reported experiencing preoperative chronic pain. The mean postsurgery assessment time for the cohort was 41.46 ± 25.10 months. At this time, 27.8% (N = 176) of all patients reported CPSP at the midline sternotomy site; 36 (5.68) others reported chronic pain at a site other than the midline sternotomy.Patient demographics and CPSP patient characteristics are reported in 0–10 scale), 35.8% reported moderate pain , and 17.6% reported severe pain . No sex differences in the development of CPSP were found. Results of the multivariable multinomial regression model showed that patients reporting moderate or severe CPSP were younger (P < 0.05), had a higher BMI (P < 0.001), and had a shorter postoperative follow-up time (P < 0.001) compared to those without CPSP , with 52.3% of patients with CPSP reporting a prior history of chronic pain compared to just 37.5% for those who did not develop CPSP. This association was no longer significant in the multivariable multinomial regression model, however.Of the 258 patients who had surgery to harvest the saphenous vein, 38 (14.7%) developed chronic leg pain. Of these 38 patients, 24 (63.2%) also developed midline sternotomy CPSP. Furthermore, patients reporting previous chronic pain elsewhere in the body were more likely to have CPSP (P < 0.001), but not sex (P > 0.05), was significantly associated with CPSP status such that patients with CPSP were younger than those without CPSP. Unlike in the short survey, there were no significant differences in BMI with no CPSP as the reference category (P > 0.05). Among the psychological variables, results of the multivariable multinomial regression model showed that anxiety sensitivity was significantly higher among patients with moderate pain (P = 0.011); patients with severe CPSP had significantly higher levels of pain catastrophizing (P = 0.007) and patients with moderate or severe CPSP had significantly higher levels of somatization (P ≤ 0.001) compared to those without CPSP (Age (out CPSP .P < 0.05). There were also significant differences in the thermal pain detection thresholds such that patients with pain had lower heat and cold pain detection thresholds at the forearm (P < 0.05), thus showing cold allodynia and heat hyperalgesia  demonstrated significant differences on several noxious cold and heat assays but not on nonnoxious warm and cool psychophysical assays . Resultsralgesia .10.1080/8This is the largest postsurgical cohort study to date that reports on the pain and psychological phenotypes of patients who developed CPSP following cardiac surgery, in contrast to those not developing CPSP after the same surgery. In keeping with previous studies demonstrating CPSP rates ranging from 11% to 40% after cardiac surgery, we report that 27.7% of the patients studied in the present cohort developed such chronic pain.Of the 176 patients who reported CPSP, 46.6% reported low to mild pain, 35.7% reported clinically relevant moderate pain, and 17.6% reported clinically relevant severe pain in the operated field and the nearby region of the chest. Yet only approximately 6% of the cohort developed chronic pain following a concurrent surgery to harvest the saphenous vein. It is possible that the lower incidence of chronic postsurgical pain in the calf scar is due to the smaller extent of tissue and nerve injury or the use of a pre-emptive peri-operative use of local anesthesia. It is also possible that the use of special spanners to enable access to the heart for the duration of the surgery caused compression of intercostal nerves and added causes for increased rates of CPSP compared to calf surgery.A majority of patients (63.2%) who developed chronic pain in the calf also developed sternotomy CPSP. Likewise, many of those with CPSP reported a prior history of chronic pain elsewhere in the body. These associations may suggest that chronic pain is an innate trait, controlled by the same genetic variants that express their potential whenever there is surgery. These findings are also compatible with the strong tendency to develop the same chronic pain if amputated twice in life, either concurrently or delayed or years apart.18 Depression and emotional numbing did not show a significant difference in reporting between the two groups unlike previous work.13 However, our study is congruent with previous work demonstrating that the transition to CPSP is associated with pain rumination,20 the tendency to catastrophize,20 pain magnification,20 functional disability,5 fear of pain and anxiety,21 and younger age.24In accordance with previous literature, patients presenting for surgery with prior chronic pain were more likely to develop CPSP.25 patients with CPSP are at risk of psychological diathesis from sensitivity to bodily sensations26 and anxiety sensitivity.27 With regards to the cognitive–behavioral fear-avoidance model, responses of patients with CPSP to psychological questionnaires indicate a greater likelihood of being fearful and avoidant of pain.29 Finally, this patient population reports increased pain catastrophizing compared to patients without chronic pain. Such individuals are likely to engage in catastrophic thinking if they are genetically predisposed to respond fearfully to pain and if they show signs of posttraumatic stress.30 Recent studies highlight the importance of assessing posttraumatic stress symptoms and their relevance to pain chronicity in patients undergoing major surgery.31 For these patients, when pain is perceived as a threat, a vicious cycle is initiated in which pain-related catastrophizing and anxiety dominate the patient’s experience. This leads to somatic hypervigilance, activity avoidance, and disability, which in turn feed back into the pain experience to fuel pain-related fears.6 Alternatively, it is possible that the same genes that predispose patients to transition to chronic pain due to a psychological vulnerability such as depression, anxiety, and catastrophizing may have a pleiotropic role in peripheral and/or central pain pathways. This could predispose patients to develop abnormal hyperexcitability in injured primary afferents and/or in central nerve system (CNS) networks that abnormally process afferent nociceptive inputs. An example of such a gene is COMT, polymorphisms in which have been associated with catastrophizing, anxiety, and fear (by operating in the CNS), and the same polymorphisms concurrently affect breakdown of adrenaline and noradrenaline in sympathetic efferents in injured nerve-end neuromas,32 where they may mediate sympathetically maintained pain.Based on existing validated models of chronic pain and disability ,Though patients with CPSP developed cold and hot allodynia in the forearms bilaterally and cold hyperalgesia in the arm and hand, no such abnormalities were detected in the chest. Regrettably, we did not use the quantitative sensory testing (QST) assay to test for these sensory abnormalities at the calf. However, in contrast to abnormalities at the noxious intensity range, patients with CPSP did not develop cool or warm hyperesthesia; that is, there were no differences between the groups with regards to detection thresholds of nonpainful sensations of cool or warm in the forearms.Using mechanical algometry, we found that having CPSP was associated with a trend for mechanical allodynia in the sternum. Because patients were instructed to press a button to stop the further increase in stimulus intensity beyond the pain threshold, we do not know whether they also developed mechanical hyperalgesia. Not withstanding, based on the thermal sensory abnormalities discovered in this study, we can conclude that spontaneous CPSP at the field of sternotomy and cardiac surgery involves long-lasting sensitization of pain (but not of nonnoxious) pathways in the CNS to natural stimuli applied both intra- and extraterritorially. Sensory abnormalities within the region innervated by nerves injured during surgery are defined here as intraterritorial abnormalities, whereas extraterritorial abnormalities appear outside the field innervated by nerves injured during the surgical process.central sensitization, whereby there are changes to the processing of sensory nociceptive CNS inputs, rather than changes in the periphery.33 Under normal conditions, many projection neurons in the spinal dorsal horn that are driven by nociceptive input also receive converging nociceptive and nonnociceptive inputs from receptive fields innervated by neighboring nerves. When activated by natural stimuli, these convergent afferent inputs from surrounding receptive fields are normally inhibited and unable to effectively drive those projection neurons. Thus, stimulation of the forearms and hands cannot normally activate CNS projection neurons that receive their main nociceptive input from the sternal and cardiac afferents and vice versa. This is at the heart of the high resolution of nociceptive and nonnociceptive somatotopic maps that process sensory inputs from all modalities. But after peripheral nerve injury (as occurring in midsternotomy and cardiac surgery), there are long-lasting changes to spinal segmental, intersegmental, and inhibitory pathways descending from supraspinal structures in the CNS. When disinhibited, spinal projection neurons become hyperexcitable and respond more vigorously to afferent nociceptive and nonnociceptive inputs from the center of their receptive fields, causing allodynia, hyperalgesia, and spontaneous pain.One potential explanation is that signals stemming from injury may reach the somata of neighboring intact primary afferents to trigger long-term changes in the expression profile of genes encoding proteins engaged in their excitability . However, evidence supports an alternative explanation termed 35 Another plausible explanation for the above finding could be that the increased pain is due to deep inspiration exacerbating their chest wall nociceptors rather than from sympathetic input or a combination of the two mechanisms.This study additionally found that CPSP is associated with significantly increased spontaneous pain, evoked by an inspiratory gasp. This maneuver is known to activate a transient increased sympathetic tone throughout the body. This increase in spontaneous pain can be explained by the documented upregulation of the expression of genes encoding components of adrenoreceptors and their shipment downstream by axonal transport and assembly in the peripheral ends of injured afferents. It is here that the upregulated adrenoreceptors are excited by circulating adrenaline and noradrenaline released from postganglionic sympathetic efferents terminating in the injured nerves.36 We cannot explain this result.Surprisingly, we found that patients with CPSP have a lower baseline systolic blood pressures compared to patients without CPSP, suggesting that CPSP is associated with a decreased sympathetic tone at rest. This finding is counterintuitive because the presence of inescapable and undertreated pain is known to be associated with stress, depression, and anxiety.37 A higher BMI increases the technical difficulty of cardiac surgery and may expose such patients to prolonged retraction and a higher incidence of chronic pain. Further, we did not find between-sex differences in our study. Though chronic pain is reported to be more prevalent in females, studies of chronic pain after hernia surgery do not find an association between sex and chronic pain.40 Of note, Taillefer et al. did not find sex to be a predictor of chronic pain after cardiac surgery.41Finally, we found an association between patients with obesity (BMI > 25) and CPSP. This confirms the findings of Bruce et al., who reported that patients who were overweight or obese at the time of cardiac surgery were more likely to report chronic pain.These results highlight that there are distinct physiologic differences in patients who have developed CPSP and those that who have not. Because this was a cross-sectional study, we are unable to comment on temporal changes in psychophysical testing. Further prospective, longitudinal investigations should seek to delineate this time course and the critical time points that differ as patients develop CPSP whereas others do not.Because CPSP cannot be cured nor prevented and available mediations do not provide sufficient pain relief, there is a need for novel treatment targets, which are expected to originate from pharmacogenomics. This solution will become especially effective because current estimates suggest that heritability of chronic pain ranges from 30% to 70%.Several challenges and considerations exist in the path forward in identifying novel CPSP genes for pharmacogenetic therapy.First, the simplistic phenomic approach of having/not having CPSP likely does not reflect the heterogeneous nature of traits contributing to the expression of CPSP. Thus, treatments based on genes found using the arch-phenome of having/not having CPSP are expected to be less effective than those tailored based on the repertoire of symptoms and signs displayed by an individual patient. To accomplish this goal would necessitate using those secondary traits as phenomes for genetic analysis, because each of these CPSP-related traits is arguably controlled by unique genes.Second, using having/not having CPSP as the only phenome for genetic analysis might miss the detection of many genes, which could be identified if using more refined, mechanism-based intermediate phenomes.Third, this raises the question of how to identify the best intermediate phenomes. In the present study, we addressed this issue by first using clinical parameters of CPSP to classify patients as having/not having CPSP and then using this grouping strategy to survey potential phenomes and intermediate phenomes that could be used for genetic association. However, the final test of how effective a CPSP-related phenotype is as a phenome can only be made if the genetic association analysis identifies genetic polymorphisms of relevance to CPSP and the effect size of carrying these polymorphisms can explain a sizable portion of the heritable, allelic risk for CPSP. Thus, to assess the effectiveness of a phenome necessitates having genotypic data of a sufficiently statistically powered cohort of patients with CPSP.Phenotyping pain and psychological questionnaires and psychophysical tests highlight key differences between patients who developed chronic pain after cardiac surgery and those who had the same surgery but did not develop such chronic pain. This is the first step in a study that aims at identifying a set of phenomes to be used in genetic association analysis of CPSP. Prospective longitudinal investigations are required to fully characterize the temporal aspects of these traits and identify genetic and environmental risk and protective factors underlying the transition from an acute pain to CPSP, its maintenance, and natural resolution in some patients.


In addition, patients with higher depression levels had lower levels of social participation. The results revealed a relationship between the psychological variables, anxiety and depression, with knee pain and the onset of symptomatic OA, as well as an influence of depression levels on social participation. Improving these psychological characteristics may be useful in delaying the onset of symptomatic OA and enhancing social participation.The main objective of this research is to determine the psychosocial differences between patients with knee pain or early osteoarthritis (EOA) and healthy subjects at risk of developing osteoarthritis. The secondary objective is to determine how psychosocial factors might influence pain and social participation in patients with EOA. A cross-sectional study was performed. Participants were divided according to the presence of pain or EOA. Pain intensity both at rest and walking, psychological variables such as anxiety and depression, and social participation were evaluated. A total of 105 participants were included (64 with knee pain and 41 without pain), with a mean age of 51.42 ± 5.92 (35 men and 70 women). Patients with knee pain had higher levels of anxiety (MD = −2.35;  Osteoarthritis (OA) is a joint disorder characterized by progressive degenerative changes ,2 that mIn this regard, multiple mechanisms can contribute to the perception of pain, such as nociception, neuropathic symptoms, psychological and personality factors, genetic influences, past painful experiences, comorbid conditions, and expectations related to future pain ,7. AmongThis relationship with psychological factors and social participation has been poorly studied for ‘early OA’ (EOA), before obvious structural or degenerative changes have been established in the joint ,2,5. A bTherefore, we believe that the identification of psychosocial factors related to the onset of symptomatology in patients with EOA could be an important way to detect and prevent the development of pain and symptomatology in patients with knee OA. The main objective of this research is to determine the psychosocial differences between patients with knee pain, EOA, and healthy subjects at risk of developing OA. The secondary objective is to identify the relationship between pain, psychosocial factors, and social participation in patients with EOA.A cross-sectional study with a non-probabilistic sample was performed. The design followed the international recommendations for strengthening the reporting of observational studies in epidemiology . All theSubjects were recruited and followed at Hospital La Fe, Valencia, Spain, within the H2020 project OACTIVE. The design of the data collection protocol started in November 2017, and the recruitment and follow-up of participants started in July 2018 and lasted until February 2021.The inclusion criteria for the pain group (PG) were (a) patient age greater than or equal to 40 years who experienced pain; (b) Kellgren and Lawrence (KL) 0–1. For the no pain group (NPG), the inclusion criteria were (a) patient age greater than or equal to 40 years, without pain or any knee symptoms; (b) Kellgren and Lawrence (KL) 0–1. The exclusion criteria were the same for both groups: (a) any cognitive disability that hindered the viewing of the audio-visual material; (b) illiteracy; (c) vomprehension or communication difficulties, (d) insufficient Spanish language comprehension to follow measurement instructions; (e) the presence of any rheumatic, autoimmune, or infectious pathology.After the clinical evaluation, we reviewed the initial classification of every subject and refined it according to Luyten’s proposal for EOA classification:(a) Patient-based questionnaires: knee injury and osteoarthritis outcome score: 2 out of the 4 KOOS subscales  need to score “positive” (≤85%).(b) Patients should present joint line tenderness or crepitus during the clinical examination.(c) X-rays: Kellgren and Lawrence (KL) grade 0–1 standing, weight bearing  .We include in this section general demographic information such as gender, age, educational level, birth country, ethnicity, residency, marital status, and economic status ,18. UnhePain Intensityp <.001) and a minimal detectable change of 15.0 mm [The Visual Analogue Scale (VAS) was used to measure pain intensity before and after each treatment. The VAS is a 100 mm line with two endpoints representing the extreme states “no pain” and “the maximal pain imaginable”. It has been shown to have a good retest reliability This instrument is the most extensively used for the functional and symptomatic assessment of patients with osteoarthritis. The WOMAC questionnaire is self-administered and is used to assess patients who progress with hip and/or knee osteoarthritis. The questionnaire is a multidimensional scale composed of 24 items divided into 3 aspects: functional pain (consisting of 5 items), stiffness (2 items), and activities of daily life difficulties (17 items). Higher values mean poorer WOMAC subscales scores of pain and physical function. The Spanish version of the WOMAC questionnaire has adequate psychometric properties, presenting an index of internal consistency (a) of 0.82 for pain and 0.93 for physical function subscales .Anxiety and Depression SymptomsThe Spanish version of the Hospital Anxiety and Depression Scale was used to assess the presence of depression and anxiety symptoms in the participants . This scSocial ParticipationThe Maastricht Social Participation Profile included nine questions to determine the frequency and diversity of social involvement for older people with chronic diseases . The following nine items comprised social participation: ‘How often in the past four weeks have you (taken part in/been to): (1) a club, interest group or activity group, church, or other similar activity; (2) a cultural or educational event such as the cinema, theatre, museum, talk, or course; (3) eaten out; (4) gone out to a pub, café, or tearoom; (5) a public event; (6) an organized games afternoon or evening; (7) a day trip organized by a club or society; (8) committee work for a club, society, or another group; (9) any organized voluntary work? The response categories ranged from zero  to three (‘more than twice a week’). We calculated the total scores for descriptive purposes, ranging from 0 to 27 , where higher scores equated to higher social participation .An information sheet with an explanation of the procedure and an informed consent form were given to all the participants. Once the subject had read the information from the study, they were allowed to ask any questions about its nature. The subjects that agreed to participate proceeded to fill in the sociodemographic questionnaire. Self-reported measures of disability, pain, and disability self-reported variables were then assessed. Finally, a physical examination was performed, including physical tests and motor and functional tests. The study protocol lasted for approximately one hour. In order to avoid fatigue affecting the physical tests, an interval of 3 min between tests was maintained. This procedure was identical for both groups.t-test was used for the group comparisons. Cohen’s d effect sizes were calculated for multiple comparisons of the outcome variables. According to Cohen’s method, the magnitude of the effect was classified as small (0.20–0.49), medium (0.50–0.79), or large (0.80).The sociodemographic and clinical variables of the participants were analyzed. The data were summarized using frequency counts, descriptive statistics, summary tables, and figures. The data analysis was performed using the Statistics Package for Social Sciences . The categorical variables are shown as frequencies and percentages. The quantitative results are represented by descriptive statistics , and standard deviation). For all variables, the z-score was assumed to follow a normal distribution based on the central limit theorem because all the groups had more than 30 participants . StudentThe relationships between pain and disability measures, as well as between physical measurements, were examined using Pearson’s correlation coefficients. A Pearson’s correlation coefficient greater than 0.60 indicated a strong correlation, a coefficient between 0.30 and 0.60 indicated a moderate correlation, and a coefficient below 0.30 indicated a low or very low correlation .A total of 105 participants were included in the study, with a mean age of 51.42 ± 5.92 (35 men and 70 women). All the participants were finally enrolled and analyzed .n = 64) and NPG (n = 41). The mean age for the PG was 51.77 ± 5.67 and 50.29 ± 6.38 for the NPG. There were no statistically significant differences between the groups in terms of age, gender, marital status, educational level, toxic habits, or hormonal status  and social participation (5.89 ± 3.44 for PG and 5.68 ± 2.73 for NPG). However, statistically significant differences were found for anxiety  and depression symptoms , with both showing higher values in the PG  and healthy subjects (HS) (n = 41). The mean age for EOA was 51.85 ± 5.72 and 50.49 ± 6.21 for HS. There were no statistically significant differences between the groups in terms of age, gender, marital status, educational level, toxic habits, or hormonal status.Participants were divided into two groups based on the EOA criteria: EOA  and social participation (5.89 ± 3.23 for EOA and 5.73 ± 3.14 for HS). However, statistically significant differences were found for anxiety  and depression symptoms, with both showing higher values in patients with EOA . Additionally, VAS showed higher values for the EOA group, and statistically significant differences were also found for VAS rest  and VAS walking   .n = 64) and another with lower levels of depression (n = 51). There were no statistically significant differences between the groups in terms of age, gender, marital status, educational level, toxic habits, or hormonal status.Differences between participants were analyzed according to their level of HAD depression. For this purpose, the median (median = 2) was calculated, and the participants were divided into a group with higher levels of depression (p = 0.004) of patients with EOA was found in the higher depression group (n = 37) compared to the HS group (n = 20). Similarly, the number of participants with lower levels of depression was lower in the EOA group (n = 17) compared to the HS group (n = 30).A statistically significant higher number . However, statistically significant differences were found for VAS rest, showing higher values in patients with higher depression values , VAS walking , and social participation, showing that patients with higher depression levels had lower levels of social participation   .p < 0.05). In addition, higher anxiety levels were correlated with higher VAS at rest  and walking , but were not correlated with WOMAC levels and social participation (Correlation analysis showed that higher levels of depression were correlated with higher levels of VAS at rest and walking, higher WOMAC, and less social participation, with a moderate correlation strength (r = 0.21 to 0.41; cipation .The main objective of this research was to determine the psychosocial differences between patients with symptomatic EOA, asymptomatic EOA, and HS at risk of developing OA. Firstly, the results showed that patients with knee pain had higher levels of anxiety and depression, regardless of the presence of EOA. No differences were found in the WOMAC values, suggesting that psychological variables could have a stronger relationship with the occurrence of pain.Knee pain is the primary complaint of those with knee OA and is associated with worse physical function, quality of life, and periarticular knee muscle function ,28. In tAmong these factors, the presence of anxiety or depression has been linked to a greater perception of pain and a lower quality of life . This isOur secondary objective was to determine how psychosocial factors might influence pain and social participation in patients with EOA. In this regard, our results showed that social participation was not significantly influenced by pain or EOA. However, statistically significant differences were found among participants with higher levels of depression. Social participation is a frequently overlooked variable, yet it has previously been associated with higher mortality rates . PreviouFinally, our data show that OA or pain could be less relevant factors than depression levels in relation to social participation, which underlines the need to include psychological interventions focused on reducing depressive symptoms in these patients from the beginning of the disease. Furthermore, they underline the need to rethink the clinical relevance of EOA. These results suggest that it may be more meaningful to assess pain or psychosocial factors in order to achieve better diagnostic and prognostic results. Further research on this matter is needed.This study has some limitations that must be considered. The cross-sectional nature of this study makes it impossible to establish causality. Longitudinal studies are needed to reveal whether psychosocial factors are a cause or a consequence of knee pain in OA. In addition, it would have been interesting to assess more psychological variables as cognitive, emotional, and emotive factors may be related to the onset of symptoms in EOA and play a role in social participation outcomes . MoreoveThe results of our study revealed a relationship between psychological variables, anxiety and depression, with knee pain and the onset of symptomatic OA. In addition, our data show that depression levels could be a relevant factor in relation to social participation in patients with EOA.


Does a virtual reality (VR) intervention compared with standard care improve pain and anxiety outcomes among patients undergoing peripheral intravenous catheter (PIVC) placement in the pediatric setting?In this randomized clinical trial of 107 patients undergoing PIVC placement in 2 pediatric clinical settings plus their caregivers and clinicians, a VR intervention significantly reduced mean patient-reported post-PIVC anxiety and pain, caregiver-reported perceptions of patients’ pain, and clinician-reported perceptions of patients’ pain and anxiety.In this study, a VR intervention significantly decreased pain and anxiety among patients undergoing PIVC placement in the pediatric setting. This randomized clinical trial examines the effect of a virtual reality intervention vs standard care on pain and anxiety associated with peripheral intravenous catheter placement among patients receiving care in 2 pediatric clinical settings. The inclusion of digital therapeutics  for the management of pain and anxiety associated with routine acutely painful medical procedures may have a substantial impact on treatment adherence and improve long-term health outcomes among young patients.To determine whether a VR intervention decreases pain and anxiety among patients undergoing peripheral intravenous catheter (PIVC) placement compared with standard care in the pediatric setting.This randomized clinical trial was conducted from April 12, 2017, to July 24, 2019, among 107 patients aged 10 to 21 years who were undergoing PIVC placement in 2 clinical settings (a radiology department and an infusion center) at an urban pediatric academic medical center in the US. Patients, caregivers, and clinicians completed pre-PIVC and post-PIVC placement questionnaires measuring patient pain, anxiety, and anxiety sensitivity; only participants with complete data from before and after PIVC placment were included in the analyses.Patients were randomized to receive standard care  or a VR intervention using a balanced computer-generated randomization scheme stratified by sex. All patients who received the VR intervention were offered concurrent standard care; however, VR plus standard care was not specifically examined.Primary outcomes were patient pain  and anxiety  reported by the patient, caregiver, and clinician after PIVC placement. Outcomes were analyzed using generalized linear modeling with backward stepwise selection for final model building.P < .001) and clinician-reported . Patients in the VR group vs the standard care group also had significantly lower mean post-PIVC pain scores when patient-reported , caregiver-reported , and clinician-reported . Aside from lower levels of baseline pain and anxiety, no demographic variables among patients in the VR group were associated with lower levels of post-PIVC pain and anxiety.A total of 107 patients  completed the clinical trial; 54 patients received standard care, and 53 patients also received the VR intervention. Patients who received the VR intervention compared with standard care had significantly lower mean post-PIVC anxiety scores when patient-reported (1.85 points  vs 3.14 points ; In this randomized clinical trial, patients undergoing PIVC placement who received a VR intervention experienced significantly less anxiety and pain compared with those who received standard care. The use of patient, caregiver, and clinician data provided a variety of subjective information, as well as observable and objective data regarding perceived pain and anxiety beyond patient reporting alone.CHLA-15-00549ClinicalTrials.gov Identifier:  The VR experience capitalizes on multisensory integration to transport the individual into computer-simulated worlds. It has also been asserted that immersive VR environments may provide more effective therapeutic interventions than standard practices  for the management of procedural pain and anxiety because of the combination of attentional demands and other cortical systems involved in VR gameplay.2 However, the heterogeneity of VR systems and environments has produced mixed scientific findings stemming from the inability of evidence-based evaluations to keep pace with continuously evolving technological innovations.4 Although there has been substantial expansion in the use of VR interventions in health care settings, the scientific evidence continues to lag behind advancements in technologies and software; relatively little is known regarding which patients may benefit from VR interventions and which procedures are best suited for incorporating VR systems.Investigators have experimented with advancements in commercial VR headsets  and mobile VR capabilities , leading to more affordable and feasible clinical trials examining the use of VR systems in health care settings.5 This pain and distress reduces treatment satisfaction for the patient, caregiver, and clinician. Furthermore, routine procedures may induce adverse emotional or traumatic reactions, especially among patients with chronic conditions receiving medical treatment that may constitute frequent vascular access.6 Given the potential detriment of painful routine medical procedures for the patient, caregiver, and clinician, it is important to consider alternative strategies for the management of pain and anxiety beyond standard care. Standard care for the management of acute procedural pain in the pediatric setting includes simple distraction techniques  and the application of numbing cream.9 A 2008 survey of child life specialists found that VR intervention was one of the strategies used least frequently for pediatric pain management,10 even though a randomized clinical trial found that VR was an effective intervention for this purpose.11 An abbreviated Cochrane review similarly found that, across 28 randomized clinical trials of psychological interventions for pain management, nonpharmacological distraction interventions were among the most beneficial in supporting the reduction of pain and distress among children undergoing needle-related procedures.12Young patients who routinely undergo painful medical procedures, such as peripheral intravenous catheter (PIVC) placement or blood sample collection, often experience pain and distress, thereby increasing their risk of needle phobia, anxiety, and treatment nonadherence.20 These VR interventions can also be implemented within a health care setting at low cost21 and have been found to offer superior pain reduction for children compared with standard care during wound dressing changes22 and IV placements before computed tomographic scans.17Virtual reality interventions have been reported to decrease pain during medical procedures, such as burn and wound care, port access, blood sample collection, intravenous (IV) placement and therapy, and dental procedures.23 Research suggests that negative emotions, such as anxiety, may facilitate pain, making anxiety an important area to consider for pain management.24 Gold et al17 found that pediatric patients receiving standard care during IV placement experienced a 4-fold increase in affective pain compared with patients receiving a VR intervention, highlighting the association between pain and anxiety among young patients undergoing routine medical procedures. Therefore, it is important to examine the association between acute pain and anxiety sensitivity, which refers to one’s propensity to be afraid of anxiety-related sensations.5The use of VR systems has also been found to reduce reported symptoms of acute anxiety during medical procedures.27 Chan et al25 examined VR use in the emergency department and outpatient settings among children undergoing IV cannulation or venipuncture, reporting a statistically significant decrease in perceived pain among the VR group in the emergency department. However, in the pathology setting, both the VR and standard care groups experienced an increase in pain, with patients in the VR group reporting a lesser increase in pain than those in the standard care group. Previous research on VR interventions for managing procedural pain and anxiety in pediatric settings has been limited, comprising small samples and results that have mostly been reported in technological and nonpediatric publications, thereby limiting the impact or relevance of the findings for direct clinical services.Research on the use of VR interventions has generally been conducted among small samples in 1 or 2 settings.The current randomized clinical trial examined the effectiveness of a VR intervention compared with standard care for reducing patient pain and anxiety during PIVC placement in 2 pediatric clinical settings. Within the VR group, patient characteristics were examined to identify which patients would most benefit from the VR intervention.CONSORT) reporting guideline for randomized clinical trials. The trial protocol appears in This study was approved by the institutional review board of Children’s Hospital Los Angeles. All patients provided written informed consent; for those younger than 18 years, patient assent and written informed consent from a parent or guardian were obtained. The study followed the Consolidated Standards of Reporting Trials  at an urban pediatric academic medical center  from April 12, 2017, to July 24, 2019. Research staff (including A.S.L.) identified potentially eligible patients before their outpatient appointments and approached them in the waiting area to determine their interest and eligibility. Patients were enrolled after obtaining written informed consent. After enrollment, patients and their caregivers completed a battery of questionnaires via Qualtrics on tablet computers  to assess baseline patient pain and anxiety. Using a balanced computer-generated randomization scheme stratified by sex, patients were randomized to receive either standard care (standard care group) or a VR intervention (VR group). All patients in the VR group were offered concurrent standard care ; however, many patients in the group declined standard care, and VR plus standard care was not specifically examined. Study personnel were blinded to the patient’s treatment group until baseline measures were completed. Patients in both groups had access to standard care procedures for PIVC placement . For patients in the VR group, research staff briefly explained gameplay and fitted them with a VR headset. Patients began gameplay less than 5 minutes before their PIVC placement and concluded gameplay after successful vascular access. After PIVC placement, patients, caregivers, and clinicians rated patient pain and anxiety levels during the procedure.Two mobile-based VR head-mounted displays were used. Patients used 2 different VR headsets depending on their age group (Samsung Gear VR [Samsung Electronics] for patients aged 13-21 years and Merge VR [Merge Labs] for patients aged 10-12 years). Both age groups played a multisensory  VR game . While playing the game, users traveled on a preset path through a colorful highly interactive 3-dimensional environment filled with animated landscapes, buildings, and clouds, during which the user’s gaze controlled the direction of a cannon fired to knock down teddy bears. For hygiene purposes, patients wore hospital hairnets while using the VR equipment, and all equipment was cleaned with alcohol-based sanitary wipes between patients. The VR intervention was implemented by trained and supervised research staff (including A.S.L.). Over the course of the clinical trial, we did not receive any reports of misuse or mismanagement of the VR equipment.Patients 18 years and older self-reported their demographic information, including age, educational level, race/ethnicity, and medical history . For those younger than 18 years, a caregiver reported the patient’s age, grade, race/ethnicity, and medical history.28 The FPS-R constitutes a horizontal series of 6 faces displaying a range of facial expressions, from no pain (0 points) to very much pain (10 points). Patients and caregivers were asked to point to the face that reflected the patient’s current level of pain. The FPS-R has been widely studied and found to have high reliability and validity in children aged 4 to 16 years.29 Clinicians also rated the patient’s level of pain during the procedure from 0 to 10 points.Patients and caregivers completed the Faces Pain Scale–Revised (FPS-R) to measure patient pain before and during the PIVC procedure.30 Clinicians also rated the patient’s level of anxiety during the procedure from 0 to 10 points.Patients and caregivers completed a visual analogue scale (VAS) to measure patient anxiety before and during the PIVC procedure. The VAS for anxiety consisted of a vertical image of a thermometer that shifted in color from yellow  at the bottom to dark red (accompanied by an image of a distressed face) at the top. Patients and caregivers were asked to point to the spot on the thermometer that corresponded with the patient’s level of anxiety, with the neutral face scored as 0 points and the distressed face scored as 10 points. Visual analogue scales have been found to have good reliability and validity in pediatric samples.31 This 18-item measure uses a 3-point Likert scale to assess the extent to which respondents believe the experience of anxiety will result in negative consequences . Items were summed . The CASI has been found to have high internal consistency (α = .87) and good test-retest reliability in both clinical (r = 0.79) and nonclinical (r = 0.76) samples.31Patients reported their anxiety sensitivity using the Childhood Anxiety Sensitivity Index (CASI).After the PIVC procedure, patients in the VR group completed the Gold-Rizzo Immersion and Presence (GRIP) Inventory  for continuous variables. To determine differences in baseline characteristics between groups, χ2 or Fisher exact tests were used for categorical variables, and the Mann-Whitney U test was used for continuous variables.A power analysis using preliminary data from a previous study of VR use among patients receiving phlebotomy, which was conducted by the principal investigator (J.I.G.),The current analysis focused on the effectiveness of VR with regard to reducing pain and anxiety. Primary outcomes were post-PIVC anxiety and pain scores obtained from each of the 3 raters . A secondary outcome was identification of patient factors within the VR group that were associated with better pain and anxiety outcomes. Generalized linear models were built to investigate differences between the patient groups using backward stepwise selection, allowing all variables to be entered and removed. Sex and location of care (categorized as radiology department vs infusion center) were the stratification variables; therefore, these variables were not eliminated from the models, whereas candidate variables, such as age  and anxiety sensitivity , were assessed for removal from and reentry in the models based on statistical significance. All models controlled for the patient’s pre-PIVC scores .Within each outcome, separate models for each rater were constructed. The adjusted mean post-PIVC scores for the VR and standard care groups were presented with 95% CIs. Interactions between patient group and the presence of chronic disease at baseline were explored in the patient models for each outcome to determine whether chronic disease at baseline modulated the effectiveness of the VR intervention. Secondary analyses explored which patient characteristics within the VR group were associated with decreased pain and anxiety.P values were assessed at the 2-tailed .05 significance level. Data were analyzed using SAS software, version 9.4 for Windows (SAS Institute).All A total of 273 patients were assessed for eligibility. Of those, 102 patients (37.4%) declined participation, primarily because of disinterest in receiving the VR intervention or limited time to participate in the study . OverallAmong patients, the median age was 14.7 years , and 63 patients (58.9%) were male . In totaP < .001) and clinician-reported . There was no evidence that chronic disease at baseline modulated the effectiveness of the VR intervention; therefore, the interaction between chronic disease and patient group was not included in the model.Multiple linear regression analyses indicated that patients in the VR group had significantly lower adjusted mean post-PIVC anxiety scores on the VAS compared with patients in the standard care group when anxiety was patient-reported . Older patient age (≥14.7 years vs <14.7 years) was associated with lower anxiety scores when rated by the clinician  but not when rated by the patient or caregiver , caregiver-reported , and clinician-reported . In the patient model, the interaction between chronic disease at baseline and patient group was removed from the model because there was no evidence that chronic disease modulated the effect of the VR intervention on patient post-PIVC anxiety. In the same model, older patient age (≥14.7 years vs <14.7 years) was associated with lower mean pain scores , whereas higher patient CASI summed scores (≥8 points vs <8 points) were associated with higher mean pain scores , regardless of treatment group  when the pre-PIVC pain score was greater than 0. When the patient’s pre-PIVC VAS anxiety score was less than 1.47, the adjusted mean post-PIVC anxiety VAS score was 1.15 points (95% CI 1.15-1.78) compared with those with a pre-PIVC pain score of 1.47 or greater, who had an adjusted mean post-PIVC anxiety score of 2.58 points (95% CI 1.87-3.28). In the VR group, age, location of care, sex, VR immersion (GRIP Inventory) score, and patient anxiety sensitivity (CASI) summed score were not associated with changes in outcomes  vs 2.22 points , only pre-PIVC pain and pre-PIVC anxiety scores were associated with changes in post-PIVC pain and post-PIVC anxiety scores for patients who received the VR intervention. Investigators have previously raised the question of whether young patients who have chronic health conditions and undergo more frequent PIVC procedures respond differently to VR intervention.32 Few VR studies have conducted prospective randomized clinical trials with a large cohort.34 This study is the first, to our knowledge, to prospectively investigate a large cohort across 2 clinical settings (a radiology department and an infusion center). The study’s results support the effectiveness of VR in both settings, with no significant differences between settings found, and they extend the findings reported in a 2018 study of VR use among patients receiving phlebotomy.1 Scientific investigation of VR interventions across a variety of settings is necessary and may lend support for the flexibility of VR systems as nonpharmacological interventions for pain management.This study’s findings add to the evidence-based literature indicating that a VR intervention is capable of decreasing pain and anxiety among patients undergoing painful procedures. In addition to quantitative decreases in pain and anxiety, the qualitative reports of patient, caregiver, and clinician satisfaction obtained in this study may have meaningful clinical implications. A previous study found that receipt of painful medical procedures was associated with a person’s overall perceptions of adverse childhood experiences, which in turn has been reported to have negative implications for long-term health outcomes.This clinical trial conducted a series of distinct analyses, triangulating the data around the patient using patient-reported, caregiver-reported, and clinician-reported scores for pain and anxiety. To our knowledge, these analyses have never been conducted in VR studies and reflect the discrete contributions of patient, caregiver, and clinician perspectives in assessing patient pain and anxiety. The current data suggest that the significance and extent of the effects of patient age, sex, location of PIVC procedure, VR intervention, and patient anxiety sensitivity differed among the 3 ratings of pain and anxiety. The VR intervention was beneficial in reducing pain in all 3 rating models and reducing perceived anxiety in 2 rating models (patient and clinician); however, perceived anxiety was not significantly different when reported by the caregiver. Future research would benefit from continuing to examine multirater assessments, including ratings from patients, caregivers, and clinicians. Additional objective measurement of outcomes, including video recordings of the patient experience before, during, and after medical procedures, would provide valuable data.35 Virtual reality, a nonpharmacological intervention, is an empirically supported, feasible, and cost-effective solution to managing pain and anxiety during routine venipuncture procedures in the pediatric setting. Virtual reality interventions can target the reduction of needle phobia; reduce adverse and traumatic reactions to medical procedures; improve satisfaction for patients, caregivers, and health care practitioners; and lead to improved outcomes as a result of consistent adherence to treatment. Future research on reducing pain and anxiety during medical procedures  may benefit from examining interventions that reduce or eliminate the use of medications . A movement toward nonsedating medical procedures using VR interventions may reduce many of the known adverse effects of medications and improve overall health outcomes for patients with acute and/or chronic medical needs.The data from this study support the management of pain and anxiety using a VR intervention among patients undergoing PIVC needle-related procedures in the pediatric setting and highlight the opportunity for nonpharmacological interventions. Given the ongoing opioid crisis, there is substantial concern about the role of medications in pain management.This study has limitations. The current findings support the use of a VR intervention for PIVC procedures in the pediatric setting; however, a number of issues should be considered when implementing VR within both inpatient and outpatient clinics. Although this study used a prospective randomized clinical trial design, the nature of patient-reported, caregiver-reported, and clinician-reported outcomes introduces bias and subjectivity. Because this study was unblinded and collected self-reported data, the patients, caregivers, and clinicians may have been motivated by a desire to please the investigator when completing the measures. These findings are also limited to the performance of a PIVC procedure in 1 radiology department and 1 outpatient infusion center at a single pediatric academic hospital. Although future studies would benefit from examining the impact of combined treatments , the size of the current sample did not permit this type of analysis.37 Given the rapidly evolving technological development of both VR hardware and software, the validation of a single VR system has inherent strengths and limitations. Although it is becoming increasingly feasible to integrate VR systems into clinical practice because of the reduced costs, availability of high-quality off-the-shelf technologies, and large number of VR environments, requirements remain regarding training and supervision of staff to operate the VR equipment, strict and vigilant hygiene practices based on hospital infectious disease standards, powering the equipment, and performance of routine updates on the operating systems and software. Misuse or mismanagement of the VR equipment could result in ineffective and problematic use.The VR hardware and software were consistently used across both clinical environments in this study, which established a sound and rigorous methodological approach; however, this approach limits the potential generalizability of the findings to 2 VR systems  and 1 VR experience  at 1 pediatric medical center. Although the field of digital therapeutics has lacked scientific data to understand which VR systems are best suited for specific interventions, the findings of the current study reflect the increasing body of evidence supporting the benefits of specific VR systems  for the management of pain and anxiety associated with both blood sample collection and PIVC placement.38 As VR systems continue to become more affordable, child life programs and other stakeholders may benefit from the introduction of a VR intervention into routine blood sample collection and PIVC procedures. The use of VR systems for reducing pain and anxiety during acute painful medical procedures, especially blood sample collection and PIVC placement, continues to be supported in the literature, from single case studies to larger-scale randomized clinical trials.43Although positive results have been reported regarding VR interventions for the management of acute procedural pain over the past 20 years, the costs, availability of hardware and software, and lack of resources to execute these interventions have hindered widespread use.45 Although the current study focused on the use of a VR intervention for managing acute pain and anxiety associated with PIVC placement, other researchers are focusing on virtual therapeutics as a solution for a number of health care problems. Harnessing the innovation and accessibility of VR to deliver nonpharmacological interventions for routine painful medical procedures may have substantial physiological and mental health implications.Combining technological innovation with scientific rigor to systematically evaluate the use of VR interventions to solve difficult health care issues has been challenging, but successful integration of these 2 areas could substantially improve nonpharmacological interventions for pain and procedural anxiety and/or distress. Many academic and private organizations are focused on developing hardware and software for VR and other digital therapeutics for implementation in health care settings.The results of this randomized clinical trial are statistically and clinically significant and support the role of VR systems during PIVC procedures in 2 pediatric clinical settings. A VR intervention significantly decreased pain and anxiety among patients undergoing PIVC placement in the pediatric setting.


P = 0.03) and overall survival (OS) . In the bTMB group, bTMB ≥ 6 mutations/Mb was associated with inferior PFS  and OS . In the immunotherapy section, bTMB ≥ 6 mutations/Mb was related to superior PFS  and objective response rates (ORRs) . These findings suggest that bTMB is a validated predictive biomarker for determining the clinical outcome of advanced NSCLC patients and may serve as a feasible predictor of the clinical benefit of immunotherapies (anti-PD-1 antibody) in the advanced NSCLC population in Yunnan Province.This study was designed to investigate the impact of blood tumor mutational burden (bTMB) on advanced NSCLC in Southwest China. The relationship between the tTMB estimated by next-generation sequencing (NGS) and clinical outcome was retrospectively analyzed in tissue specimens from 21 patients with advanced NSCLC. Furthermore, the relationship between the bTMB estimated by NGS and clinical outcome was retrospectively assessed in blood specimens from 70 patients with advanced NSCLC. Finally, 13 advanced NSCLC patients were used to evaluate the utility of bTMB assessed by NGS in differentiating patients who would benefit from immunotherapy. In the tTMB group, tTMB ≥ 10 mutations/Mb was related to inferior progression-free survival (PFS) (hazard ratio [HR], 0.30; 95% CI, 0.08-1.17; log-rank  Recently, immune checkpoint blockade (ICB) therapy has shown improved clinical benefits in patients with advanced non-small cell lung cancer (NSCLC) , 2. TissMore recent studies have demonstrated that blood tumor mutational burden  may be a substitution for overall neoantigen production –14. GandbTMB testing facilities (by NGS) have been established in our cancer center since 2018. The bTMB status in advanced NSCLC patients in the Yunnan Province is not clear, so we first investigated advanced NSCLC with bTMB in Yunnan Province and further determined the feasibility of bTMB as a prognostic biomarker for ICB therapy.This research included 2 sections: we first recruited 21 advanced NSCLC patients  and obtained tissue samples for retrospectively determining their tTMB status from the Yunnan Cancer hospital between February 2018 and August 2020. Second, we recruited 83 advanced NSCLC patients (70 patients receiving platinum-based chemotherapy and 13 patients receiving ICB therapy) with blood samples for retrospectively determining their bTMB status from the same center between January 2018 and August 2020. This work was performed in full accordance with the Declaration of Helsinki, and each patient provided informed consent. Protocol approval was acquired from the ethics committee of the Yunnan Cancer hospital. Advanced NSCLC patients from Yunnan Province with sufficient tissue or blood samples were considered eligible for this TMB analysis. Patients with known EGFR mutations or ALK translocations sensitive to targeted therapy or with an autoimmune disease were excluded. Twenty-one tissue samples underwent tTMB analysis, and 83 blood samples underwent bTMB analysis to obtain PFS and OS data. Finally, we collected other information, including clinical and molecular parameters.Plasma ctDNA extraction and purification [Thermo (k0782)], leukocyte enrichment from whole blood, and low-speed centrifugation for DNA extraction and quality identification (Qubit dsDNA HS Assay Kit) were performed before library construction. The ctDNA libraries were prepared by the Vazyme ND607 DNA Library Kit with unique identifiers  to tag individual DNA molecules. The extracted DNA was used for end repair and poly(A)-tail addition by KAPA enzymatic digestion. Target fragments were captured by magnetic beads, and PCR enrichment amplification was performed after hybridization.The tumor tissue DNA extraction and NGS library construction were performed following the methods described in our previous study .® HiSeq X-TEN for sequencing according to the manufacturer’s instructions. Both raw parameters were processed by using Illumina® HiSeq X-TEN, and error reads were corrected by using the hg19 reference genome. Our program KEYseq V2.0 was used to analyze these data.Captured probes were supplied by Integrated DNA Technologies (IDT) xGen Lockdown Probes (including 547 cancer-relevant genes) to evaluate the TMB, and then the probes were separately used to capture tissue gDNA and plasma ctDNA following standard protocols. Both captured libraries for tissue gDNA and plasma ctDNA were processed into the IlluminaThe TMB was calculated by summing all synonymous and nonsynonymous variants with ≥ 5% (0.5% in blood) frequency, which was demonstrated with the mutations per megabase (mut/Mb) unit, and germline variants and driver gene mutations were removed. Our bTMB algorithm contains single nucleotide polymorphism (SNP) sites and driver gene mutations filtering common untrue mutations, while synonymous mutations, small fragments and single base insertion-deletion mutations were not filtered. Our bTMB algorithm is based on the latest official standards of Foundation Medicine, a leading provider of genome sequencing analysis services. The tTMB cutoff point was 10 mutations/Mb, and the bTMB cutoff points were 6, 10, and 16 mutations/Mb based on recent studies , 13, 17.In the group of patients who did not receive ICB therapy, PFS was defined as the time from the start of TMB detection to objective disease progression or death, and this was evaluated with the Response Evaluation Criteria In Solid Tumors (RECIST) v 1.1 or death. OS was defined as the time from the start of TMB detection until death due to any cause. In the group of patients who received ICB therapy, PFS was defined as the time from the start of ICB treatment to objective disease progression or death. OS was defined as the time from the start of ICB treatment to death due to any cause. The ORR is measured as the percentage of patients with a complete response (CR) or a partial response (PR) (as defined by RECIST v1.1). Tumor assessments were performed every 3 months after the first TMB detection, and the last follow-up was on August 8, 2020.P values determined with log-rank test) were performed, and the hazard ratios (HRs) were calculated by using a Cox proportional hazards regression model. P levels < 0.05 (two-sided) were considered statistically significant. All statistical analyses were performed by SPSS 22.0  and Graph-Pad Prism version 8.0 .Chi-square or Fisher’s exact test was used to analyze the correlation between TMB and clinical data. For survival analysis, Kaplan-Meier curves  years; 7 (33.3%) females. Cohort 2 consisted of 83 patients with blood samples; median age, 56 (range 31-82) years; 30 (36.1%) females. Thirteen patients received anti-PD-1 antibodies therapy as a second-line treatment. No evident differences were found in the patient characteristics   (p = 0.04)   2. In thP levels were minimum values   (p = 0.04)   4. In thp < 0.01, vs. 14.2%; 95% CI, 0.03-1.19; P = 0.02, vs. median, 11.5; interquartile range, 5.7-15, P < 0.01, In this work, we further investigated the relationship between bTMB status and clinical benefit in advanced NSCLC patients who treated with ICB (anti-PD-1 antibody) therapy. The PFS was significantly shorter in patients with bTMB-low (< 6 mutations/Mb) than in patients with bTMB-high (< 6 mutations/Mb). The median PFS was 4.0 months and 10.0 months, respectively  and KRAS . In addition, through plasma NGS genotyping, we also identified a wide range of cancer-related genetic alterations. The landscape of genetic alterations had been shown in TP53 , KRAS  and PIK3A .We found a wide range of cancer-related genetic alterations depended on tissue NGS genotyping. The landscape of genetic alterations had been shown in In the tissue VAF subgroup, we found that the median TP53 VAF was 35.77% (range 5.06% to 42.20%) in patients with tTMB-low, while it was 28.93% (range 13.12% to 57.85%) in patients with tTMB-high. The median TP53 VAF was 14.50% (5.06% to 57.85%) in patients with adenocarcinoma and 38.27% (21.33% to 51.70%) in patients with squamous cell carcinoma. However, there were no significant differences in the tumor type and TMB level eTable 4In this work, we first examined the association between the tTMB values and advanced NSCLC patients’ clinical outcomes who did not receive ICB therapy in Southwest China. Our findings were consistent with our previous study , and tTMThis study also demonstrated that advanced NSCLC patients with bTMB-H (≥ 6 mutations/Mb) would benefit from ICB anti-PD-1 antibody) therapy. These findings are also similar to those from previous studies . However antibodyThe selection of bTMB cutoff points is still controversial , 13. GanIn our study, a comprehensive analysis of genetic alterations in NSCLC patients demonstrated that the majority of mutations appeared in TP53 and KRAS Figure 5In summary, our findings suggest that bTMB (cutoff point was 6 mutations per megabase) is a validated predictive biomarker for determining the clinical outcome of advanced NSCLC patients with chemotherapy. Using a bTMB cutoff point ≥ 6 mutations per megabase, we found that Yunnan advanced NSCLC patients who obtained an increased PFS benefit from anti-PD-1 antibody therapy. Further prospectively validated studies with large sample sizes are needed.There are three limitations to our study. Firstly, our study was retrospective research, which may limit the interpretation of the clinical results. Secondly, the small sample size may cause unavoidable selection bias and measurement bias, relatively weakening the reliability of our conclusions. Thirdly, in our study, there were no matched blood and clinical tissue samples from the same patient for TMB detection. Therefore, it may lead to a bias between tTMB and bTMB in diagnostic concordance assessment. Further prospective researches are expected.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/YZ, YH, and YM designed the study. YM, QL, YD, JC, WC, GZ, XL, HL, and LM finished the experiments. YM and YZ analyzed the parameters and wrote the research. All authors contributed to the research. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China (No. 81860513).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.