VE against oropharyngeal HPV‐16/18, HPV‐31/45 and HPV‐31/33/45 infections were 82.4% , 75.3% (95%CI: 12.7–93.0) and 69.9% (95% CI: 29.6–87.1), respectively. In conclusion, the AS04‐HPV‐16/18 vaccine showed effectiveness against vaccine and nonvaccine HPV‐types oropharyngeal infections in adolescent females up to 6 years postvaccination.We studied effectiveness of the AS04‐adjuvanted HPV‐16/18 (AS04‐HPV‐16/18) vaccine against human papillomavirus (HPV) oropharyngeal infections associated with the increase of head/neck cancers in western countries. All 38,631 resident adolescents from 1994 to 1995 birth cohorts of 33 Finnish communities were invited in this community‐randomized trial (NCT00534638). During 2008–2009, 11,275 girls and 6,129 boys were enrolled in three arms of 11 communities each. In Arm A, 90% of vaccinated girls/boys, and in Arm B, 90% of vaccinated girls received AS04‐HPV‐16/18 vaccine. Other Arm A/B and all Arm C vaccinated participants received control vaccine. All Arm A participants and Arm B female participants were blinded to vaccine allocation. Oropharyngeal samples were analyzed from 4,871 18.5‐year‐old females who attended follow‐up visit 3–6 years postvaccination. HPV DNA prevalence was determined by SPF‐10 LiPA and Multiplex type‐specific PCR. Total vaccine effectiveness (VE) was defined as relative reduction of oropharyngeal HPV prevalence in pooled Arms A/B HPV‐vaccinated females  What's new?Cervarix vaccine against oropharyngeal HPV‐16/18 infection and moderate effectiveness against non‐vaccine types HPV‐31/33/45 infection in female adolescents. The protective effects were observed up to 6 years after vaccination. These results provide further evidence that HPV vaccination holds the potential to reduce oral HPV infection, thereby offering protection against HPV‐related head and neck cancers.The incidence of oropharyngeal cancers due to human papillomavirus (HPV) infection is increasing in Western countries. However, the efficacy of HPV vaccines against oropharyngeal infection is not well documented. Based on a large community‐randomized trial in Finland, the authors report high effectiveness (82.4%) of  AlaluminumAS04‐HPV‐16/18AS04‐adjuvanted HPV‐16/18 vaccineCIconfidence intervalGCPgood clinical practiceHBVhepatitis B virusHPVhuman papillomavirushrHPVhigh‐risk human papillomavirusesIARCInternational Agency for Research on CancerMPLmonophosphoryl lipid An (%)number (percentage) of subjects reporting an eventNnumber of subjects with available resultsOPSCCoropharyngeal squamous cell cancerVEvaccine effectivenessOncogenic, high‐risk human papillomaviruses (hrHPV) cause worldwide 9% of cancers in females and 1% in males.In western countries, the incidence of HPV‐positive OPSCC has increased very rapidly, virtually doubling in a decade since the 1970s.In the adolescent and young adult populations, the prevalence of oral HPV infections varies widely from 0.4% to 10% mostly depending on the method used for viral DNA detection.All the 33 randomized Finnish communities had more than 35,000 inhabitants.clinicaltrialgov.com) and its amendment for the collection of the oral gargle samples were approved by the ethical committee of the Pirkanmaa hospital district in 2007 and 2011. The trial was conducted in accordance with good clinical practice (GCP) and all applicable regulatory requirements including the Declaration of Helsinki. Participants aged <15 years provided written informed assent; written informed consent was obtained from the participants’ parents or legal representatives. Written informed consent was obtained from study participants aged 15 years in line with local regulations.The primary HPV‐040 trial  vaccine, and 10% received Engerix B vaccine .3+). In Arm B, 90% of vaccinated females received the AS04‐HPV‐16/18 vaccine, and 10% of vaccinated females and all vaccinated males received the HBV vaccine. The 90 and 10% proportions were receiver‐blinded proportions to assess the herd effect. The blinding was maintained for girls and boys in Arm A and for girls in Arm B. In Arm C communities, all vaccinated participants received the HBV vaccine. Virtually all (99%) vaccinated participants received all three vaccine doses.The vaccination (first three study visits) took place at the healthcare facilities of 250 municipal junior high schools of the study site communities. In Arm A, 90% of vaccinated participants received Both vaccinated and nonvaccinated females of the 1994–1995 birth cohorts from the study site communities were invited to attend follow‐up visit at the age of 18.5 years during 2012–2014. Cervical and oropharyngeal samples for HPV DNA testing were obtained on each visit. Setting the age for attending the follow‐up visit at 18.5 years was per protocol to allow a minimum of 3 years between vaccination and cervical sampling.During the follow‐up visit, the attendees filled‐in a questionnaire on living conditions, life‐habits and sexual behavior. At age 18.5 years, a cross‐vaccination with either the HBV vaccine or the AS04‐HPV‐16/18 vaccine was offered to the attendees. Female attendees (139), who had moved between Arm C and Arm A or B communities during the follow‐up by age 18.5 years were removed from the final analyses.All samples were analyzed by PCR for HPV DNA. HPV typing was performed by a broad‐spectrum PCR SPF10‐LiPA25  using HPV‐specific hybridization probes enabling detection of 14 oncogenic HPV types  and 11 nononcogenic HPV types . To ensure maximum sensitivity in the detection of HPV‐16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 6 and 11, samples initially considered to be SPF‐10/DEIA positive for HPV were re‐evaluated by the multiplex type‐specific PCR.The number of subjects invited to participate in the study and the number of subjects enrolled was tabulated by gender, for birth cohorts 1994, 1995 and overall.The analysis of VE was done on female study participants and was based on the total enrolled cohort which included all study participants from all communities, including subjects who only completed the behavioral questionnaire at 18.5 years of age. For the analysis of a specific endpoint, only subjects with measured endpoints were considered.The statistical analysis of VE of the AS04‐HPV‐16/18 vaccine against oropharyngeal infection was done by comparing the prevalence in females vaccinated with the AS04‐HPV‐16/18 vaccine from pooled Arms A and B over the prevalence in all females from Arm C for birth cohorts 1994–1995, for the following HPV types: HPV‐16, HPV‐18, HPV‐16/18, HPV‐6/11, HPV‐31/45 and HPV‐31/33/45.The VE was computed as 1 minus the odd ratio of prevalence rates between the investigated arms and the control arm, estimated using the Mantel–Haenszel test adjusted for clustering and stratified by the historical seroprevalence used in the randomization . The 95% confidence interval (CI) on VE was computed using the general inverse variance approach.As a post hoc analysis, the VE of the AS04‐HPV‐16/18 vaccine against oropharyngeal infection for birth cohorts 1994–1995 was computed for the following HPV types: HPV‐31, HPV‐33, HPV‐35, HPV‐45 and HPV‐52. For this post hoc analysis, only point estimated of the VE was provided due to the low number of events. It was computed as 1 minus the prevalence , and indistinguishable between the two types . The analyses related to the present objective are descriptive only.We hereby disclose results of our trial's secondary objective that was to evaluate VE of the AS04‐HPV‐16/18 vaccine against oropharyngeal infections in females of the 1994 and 1995 birth cohorts at the age of 18.5 years. Other objectives will be described in a separate publication and are available on the Clinical Trial Register .Click here for additional data file.


Prolonged PR interval (PRi) is associated with adverse outcomes. However, PRi determinants are poorly known. We aimed to identify the clinical determinants of the PRi duration in the general population.Some clinical data are associated with prolonged PRi.Cross‐sectional study conducted between 2014 and 2017. Electrocardiogram‐derived PRi duration was categorized into normal or prolonged (>200 ms). Determinants were identified using stepwise logistic regression, and results were expressed as multivariable‐adjusted odds ratio (OR) . A further analysis was performed adjusting for antiarrhythmic drugs, P‐wave contribution to PRi duration, electrolytes , and history of cardiovascular disease.Overall, 3655 participants with measurable PRi duration were included , and 330 (9.0%) had prolonged PRi. Stepwise logistic regression identified male sex (OR 1.41 [1.02‐1.97]); aging ; increased height ; hypertension (OR 1.37 [1.06‐1.77]); and hs troponin T (OR 1.67 [1.15‐2.43]) as significantly and positively associated, and high resting heart rate  as negatively associated with prolonged PRi. After further adjustment, male sex, aging and increased height remained positively, and high resting heart rate negatively associated with prolonged PRi. Hypertension and hs troponin T were no longer associated.In a sample of the Swiss middle‐aged population, male sex, aging and increased height significantly increased the likelihood of a prolonged PRi duration, whereas a high resting heart rate decreased it. It reflects the conduction through the atria, atrioventricular (AV) node, bundle and its branches, and Purkinje fibers.The clinical determinants of prolonged PRi are mostly unknown. Positive associations between the PRi and greater age,22.1The design of the CoLaus study with the detailed baseline and follow‐up methodologies has been reported previously.2.2ECGs were digitally recorded in a resting supine position using a single device . In accordance with the local standards, paper speed was 25 mm/second and calibration 10 mV/mm. Digital ECGs were stored in an anonymised database of SEMA Data Management System .ECG measurements were determined by Schiller AG algorithms. As automated measurements of ECG intervals significantly vary between manufacturers2.3Age was categorized in four 10‐year groups .2 and overweight as BMI ≥25 kg/m2 and < 30 kg/m2. Waist circumference was measured midway between the lowest rib and the iliac crest using a non‐stretchable tape and the average of two measurements was taken. Abdominal obesity was defined as a waist circumference ≥ 102 cm (men) and ≥ 88 cm (women).Body weight and height were measured with participants barefoot and in light indoor clothes. Body weight was measured to the nearest 100 g using a Seca scale  and height was measured to the nearest millimeter using a Seca height gage. Obesity was defined as a body mass index (BMI) ≥30 kg/mAlcohol consumption and smoking status were assessed by self‐filled questionnaire. Excessive alcohol consumption was defined as >40 g/day for men and > 20 g/day for women.Cardiovascular risk assessment was evaluated with two risk equations, the European Society of Cardiology SCOREResting heart rate was obtained on the ECG and defined as high when ≥70 beats per minute.History of cardiovascular disease (CVD) included myocardial infarction, angina pectoris, percutaneous revascularization or bypass grafting, stroke or transient ischemic attack. History of CVD was obtained either based on patient's report (for some of the events occurring before the baseline CoLaus survey) or based on clinical data (obtained during follow‐up) validated by an independent adjudication committee including cardiologists and a neurologist.Participants listed their medications in the self‐filled questionnaire. Antiarrhythmic drugs including digoxin, calcium channel blockers (CCBs), amiodarone, and beta‐blockers were selected for adjustment because of their impact on the PRi.2.4Fasting venous blood samples were processed in the Lausanne University Hospital laboratory. Biological parameters included glucose; HbA1c; total, HDL and LDL‐cholesterol; triglycerides; creatinine; NT‐proBNP; high‐sensitivity cardiac troponin T (hs cTnT), and electrolytes  for their effect on cardiac conduction.2 (1 mL/s/m2) using the CKD‐EPI formula. Dyslipidemia was defined either by using the LDL‐cholesterol thresholds adapted from the Systematic Coronary Risk Evaluation (SCORE) risk charts (Table Diabetes mellitus was defined as fasting plasma glucose ≥7.0 mmoL/L and/or HbA1c ≥48 mmol/mol (≥6.5%) and/or anti‐diabetic treatment. Renal failure was defined by eGFR <60 mL/min/1.73 mts Table , and/or 2.5Exclusion criteria for the current analyzes were as follows: (a) uninterpretable ECG ; (b) no sinus rhythm or paced rhythm; (c) Wolff‐Parkinson‐White syndrome or ≥ second degree AV block; and (d) missing phenotypic data Figure .2.6Statistical analyzes were conducted using STATA version 15.1 for Windows . Concordance between automatic and manual PRi measurements was assessed by Spearman correlation and Lin's concordance coefficients.t‐test for continuous variables. Results were expressed as number of participants (percentage) or as average ± SD. Multivariable analysis using the PRi duration as dependent variable was performed by stepwise forward logistic regression and findings were further confirmed by stepwise backward logistic regression. Results were expressed as odds ratio (OR) and 95% confidence interval (CI).Bivariate analysis of the factors associated with prolonged PRi was performed using chi‐square for qualitative variables and Student's Model 1 tested the following covariates: sex; age ; height (continuous); BMI ; waist ; alcohol intake ; smoking status ; 10‐year risk of coronary heart disease (CHD) ; diabetes mellitus (yes/no); hypertension (yes/no); dyslipidemia (yes/no); renal insufficiency (yes/no); resting heart rate ; hs cTnT  and NT‐proBNP . Model 2 tested the same set of variables as model 1, but adjusting for antiarrhythmic drugs; electrolytes ; P‐wave contribution to the length of the PRi ; the approval was renewed for the first  and the second  follow‐up. The study was performed in agreement with the Helsinki declaration and the applicable Swiss legislation. All participants gave their signed informed consent before entering the study.33.1P < .001). The digital algorithm related to the PRi was incorrect for two ECGs: (a) the digital diagnosis was sinus rhythm with an extremely long PRi, while the correct manual diagnosis was AF; (b) the digital diagnosis was an irregular rhythm with no P‐wave detected, while the correct manual diagnosis was sinus rhythm. Furthermore, P‐wave and PR values were missing in a correctly diagnosed case of sinus bradycardia.Spearman's rho was 0.95 and concordance correlation coefficient 0.95  were excluded. The reasons for exclusion are shown in Figure 3.3Of the 3655 participants with interpretable ECG and measurable PRi duration, 330  presented with a prolonged PRi. The clinical characteristics of the participants, overall and according to categories of PRi duration, are presented in Table Table After further adjustment according to model 2, male sex, older age, and increased height remained positively, and high resting heart rate negatively associated with prolonged PRi. Conversely, hypertension and hs cTnT were no longer associated. Results were similar after exclusion of participants under beta‐blockers and non‐cardioselective CCBs (model 3).3.4The results of the sensitivity analysis using inverse probability weighting are summarized in 4In this study, male sex, older age and increased height were significantly and positively associated with prolonged (>200 ms) PRi, while high resting heart rate was negatively associated. These associations were independent of the P‐wave contribution to the length of PRi.4.1The concordance between manual and digital measures of PRi duration and PRi‐related diagnoses was good. It has been demonstrated that errors in digital ECG diagnoses are frequently related to arrhythmia and conduction disorders.4.2In our sample, approximately one out of 11  participants had a prolonged PRi. This is in mid‐range of other studies reporting prevalence rates ranging from 1.6% to 18%.4.3Older age was positively associated with prolonged PRi, participants aged >75 years having a more than fourfold increase in the likelihood of prolonged PRi compared to the youngest age category. Similar findings were obtained when age was used as a continuous variable. This is a consistent finding in the literature.Male sex was positively associated with prolonged PRi, a finding also reported elsewhere.A 5 cm increase in height increased the likelihood of a prolonged PRi by 26%. To our knowledge, height has been seldom associated with ECG characteristics. Nonetheless, both the PRi and height have been linked with AF,High resting heart rate was the only factor associated with a reduced likelihood of a prolonged PRi, a finding also reported elsewhere.Hypertension and elevated hs cTnT were positively but inconsistently associated with prolonged PRi. A possible explanation for hypertension not being retained in model 2 is linked to the adjustment for medication. As hypertension was defined partly by the presence of antihypertensive drugs (beta‐blockers and CCBs included), adjusting for antihypertensive drugs reduced the strength of the association. Still, hypertension was not retained even after excluding participants on beta‐blockers and non‐cardioselective CCBs. This echoes the contradictory findings of the literature, where significant4.4This study has several limitations. First, it was limited to an age range of 45 to 86, and might not be applicable in younger or older participants. Second, the sample was mostly restricted to Caucasians and might not be generalizable to other ethnicities. Third, a sizable fraction (one‐quarter) of the sample was excluded, and excluded participants differed from the included ones regarding the levels of several determinants of prolonged PRi; this might have biased the associations between potential determinants and prolonged PRi. Still, the results obtained were almost identical when inverse probability weighting was applied. Finally, most PRi durations were digitally measured and errors may have occurred. However, we endeavored to control the reliability of the digital analyzes and optimize the manual reading.4.5In a sample of the Swiss middle‐aged population, male sex, older age, and increased height significantly increased the likelihood of a prolonged PRi duration, whereas high resting heart rate decreased it. The effect of hypertension and elevated hs cTnT on the PRi duration needs further investigations.The authors declare no potential conflict of interest.M.B., P.M.V., P.V. and J.S. designed the present study; all authors were involved in data collection; M.B. drafted the manuscript; P.V., P.M.V., F.B., E.P and J.S. critically revised the manuscript. All authors gave final approval.The funding source was not involved in the study design, data collection, analysis and interpretation, writing of the report, or decision to submit the article for publication.Table S1 LDL cholesterol levels considered for the definition of dyslipidemia, according to total cardiovascular risk (SCORE).s2: Characteristics of excluded and included participants, CoLaus/PsyCoLaus, Lausanne, Switzerland, 2014‐2017.Table s3: Sensitivity analysis conducted using inverse probability weighting, CoLaus/PsyCoLaus study, Lausanne, Switzerland, 2014‐2017.Table 4: Sensitivity analysis conducted using inverse probability weighting with age and heart as continuous variables, CoLaus/PsyCoLaus study, Lausanne, Switzerland, 2014‐2017.Table SClick here for additional data file.


We determined the association between ratios of plasma ceramide species of differing fatty‐acyl chain lengths and incident dementia and Alzheimer’s disease (AD) dementia in a large, community‐based sample.We measured plasma ceramide levels in 1892  dementia‐free Framingham Offspring Study cohort participants between 2005 and 2008. We related ratios of very long‐chain  to long‐chain (C16:0) ceramides to subsequent risk of incident dementia and AD dementia. Structural MRI brain measures were included as secondary outcomes.P = 0.056). Higher ratios of ceramides C24:0/C16:0 and C22:0/C16:0 were also cross‐sectionally associated with lower white matter hyperintensity burden on MRI , but not with other MRI brain measures.During a median 6.5 year follow‐up, 81 participants developed dementia, of whom 60 were diagnosed with AD dementia. In multivariable Cox‐proportional hazards analyses, each standard deviation (SD) increment in the ratio of ceramides C24:0/C16:0 was associated with a 27% reduction in the risk of dementia  and AD dementia . The ratio of ceramides C22:0/C16:0 was also inversely associated with incident dementia , and approached statistical significance for AD (HR 0.73, 95% CI 0.53–1.01, Higher plasma ratios of very long‐chain to long‐chain ceramides are associated with a reduced risk of incident dementia and AD dementia in our community‐based sample. Circulating ceramide ratios may serve as potential biomarkers for predicting dementia risk in cognitively healthy adults. Discovering novel blood‐based biomarkers for dementia can identify disease at an early preclinical stage, serve as surrogate outcomes for clinical trials of investigational therapies and inform our understanding of the underlying biological pathways of disease onset and progression. Altered lipid metabolism is believed to play an important role in the development of dementia and Alzheimer’s disease (AD).Recently, attention has focused on the role of circulating ratios of a very‐long‐chain to long‐chain ceramides as biomarkers for major vascular eventsn = 5124), 3021 were alive and attended examination eight, 2008 of whom were aged 60 years or above, confirmed free of dementia and with available dementia status on follow‐up. Of these, 1892 had plasma ceramides measured at examination eight, resulting in the sample for the present investigation  and had available plasma ceramide measurements. For analyses of amyloid‐burden on brain‐PET, we included participants in the dementia cohort who had available PET data at exam nine (n = 48).The Framingham Offspring cohort is a longitudinal epidemiological study of individuals recruited between 1971 and 1975. This large, predominantly middle‐aged cohort has been prospectively followed for the development of vascular risk factors, decline in cognitive function, dementia and stroke for over 45 years.Our primary outcome measure was incident all‐cause dementia developing at any time after the 8th examination cycle and up to the end of 2016. From examination cycle five (1991–1995) onward, all participants in the Offspring cohort were systematically screened using the Mini‐Mental State Examination (MMSE) and annual health status updates for the development of dementia. From examination cycle seven onward, we invited all participants to complete neurocognitive testing. We completed more detailed cognitive testing in those cases where the participant’s MMSE score was lower than the education‐based cut‐off, three points lower than the preceding examination, or five points lower than the previous highest recorded score or if the participant, family member, or Framingham study physician was concerned about cognitive impairment.11‐C PiB as a secondary outcome measure in exploratory analyses (see Appendix for further details).We evaluated structural MRI brain measures in secondary analyses, including total parenchymal brain volume (TBV), hippocampal volume (HV), white matter hyperintensity volume (WMHV) and covert brain infarcts (CBI). MRI imaging was performed using a Siemens Avanto 1.5 Tesla machine. More detailed methods on MRI brain measures have previously been published.Fasting blood samples were drawn from participants in the early morning, centrifuged and stored at −80°C until assays were performed. A modified and fully validated two‐dimensional liquid chromatography‐tandem mass spectrometry (LC–MS) assay was used to quantify plasma levels of ceramides C24:0, C22:0, and C16:0 according to previous published methods.We adjusted for baseline demographics and covariates (measured at examination cycle eight), which have previously been associated with plasma ceramide ratios and risk of dementiaWe standardized ceramide values to facilitate comparisons as continuous variables. Multivariable Cox proportional hazards models were estimated to evaluate the association between circulating ceramide ratios and risk of incident all‐cause dementia and AD dementia. Participants were followed from the baseline examination cycle eight to the time of the incident event. For individuals without incident events, follow‐up was censored at the time of death or the date the participant was last known to be dementia free, through December 2016. The assumption of proportionality of hazards was confirmed, and results are reported as hazard ratios (HR) with corresponding 95% confidence intervals (CI). Model one adjusted for age and sex, and model two additionally adjusted for education, systolic blood pressure, use of antihypertensive medication, prevalent CVD, ApoE4 carrier status; TC:HDL ratio, use of lipid‐lowering therapies, and serum TG.. We also completed a sensitivity analysis excluding those with a history of prior stroke (n = 52). Our primary, a priori, analyses evaluated the associations between the ratio of ceramides C24:0 to C16:0 and C22:0 to C16:0 and risk of incident dementia. In secondary (exploratory) analyses, we related plasma concentrations of individual ceramide species  to the risk of incident dementia and AD dementia prospectively, and with structural MRI brain measures, cross‐sectionally. In addition, we estimated multivariable linear regression models to relate plasma ceramide ratios and species to amyloid‐PET burden, adjusting for age, sex, and time from blood draw to PET scan. A two‐sided P < 0.05 was considered statistically significant. We completed all analyses using SAS version 9.4 .We estimated logistic (for binary outcomes) and linear (for continuous measures) regression models to evaluate the cross‐sectional associations between circulating ceramide ratios and MRI‐based structural brain measures, including CBI, WMHV, TBV, and HV. Models were adjusted for age, age squared (given age and brain volume show a nonlinear association), sex, time from blood draw to MRI brain, use of antihypertensive medication, systolic blood pressure, prevalent cardiovascular disease, TC:HDL ratio, use of lipid‐lowering therapies, and serum TG. We investigated for interactions between ceramide ratios and sex, ApoE4 carrier status and TC:HDL‐C ratio (<4:1 vs. ≥4:1) for the primary outcome of incident dementiaOur sample included 1892 eligible participants. The mean age of the cohort was 70.1 (SD 6.9) years and 54% were women. Baseline characteristics are shown in Table P = 0.023) and AD dementia . The ratio of ceramides C22:0/C16:0 was also inversely associated with incident dementia , but not AD dementia  (Table During a median follow up of 6.5 (IQR 5.5–7.7) years, 81 individuals were diagnosed with dementia, 60 of whom had AD dementia. In multivariable Cox‐proportional hazards regression models adjusted for demographics, vascular risk factors, use of lipid‐lowering therapies and ApoE4 carrier status, the ratio of ceramides C24:0/C16:0 was inversely associated with incident dementia   Table .P = 0.01) but not overall dementia . None of the other ceramide species was individually associated with dementia risk  and AD dementia (C24:0/C16:0) prospectively, as well as with a lower burden of white matter disease on MRI brain and B‐amyloid burden on PET (C24:0/C16:0).Previous studies have reported conflicting findings regarding the association between individual plasma ceramide species and the risk of AD dementia. Some studies have reported an association of both elevated long‐chain ceramides C16:0)6:01, 14,Individual ceramide species are believed to have different functions according to their length of fatty acyl chain. While very long‐chain fatty acyl ceramides are important for myelin function and have been suggested to protect against dementia,β‐cell dysfunction, insulin resistance and vascular reactivity,Recent cardiovascular studies have consistently shown an increased risk of adverse vascular events and mortality in individuals with increased ratios of long‐chain to very long‐chain ceramides in plasma.n = 48), an elevated ratio of C24:0 to C16:0 was associated with reduced ß‐amyloid burden on PET‐brain. Ceramides have been shown to stimulate ß‐amyloid formation, the pathological hallmark of AD, through regulating ß‐site amyloid precursor protein cleaving enzyme 1 (BACE‐1) activity,In a small exploratory analysis within our cohort  with a modest number of outcome events and the need for validation of these findings in other cohorts. Furthermore, blood ceramide concentrations may change over time and we were unable to assess stability of the ratios during follow‐up via repeat measurements. In addition, the Framingham Offspring Study is predominantly white and this will limit the generalizability of our findings to other races/ethnicities.To our knowledge, this is the first study to demonstrate that higher ratios of very long‐chain to long‐chain ceramides in the blood are associated with a reduced risk of incident dementia and AD dementia. Our study provides evidence supporting the hypothesis that individual ceramide species have differing biological roles in the pathophysiology of dementia. If our findings are confirmed, circulating ratios of very long‐chain to long‐chain ceramides may be useful biomarkers for predicting future dementia risk among cognitively healthy adults, several years in advance of clinically evident disease.Study concept and design: McGrath, Seshadri, Himali, Vasan. Acquisition, analysis, or interpretation of data: All authors. Drafting of the manuscript: McGrath. Critical revision of the manuscript for important intellectual content: All authors. Statistical analysis: Himali. Obtained funding: Seshadri, McGrath. Study supervision: Seshadri.None to report.Data S1. Supplementary methods.Table S1. Ceramides and amyloid burden on PET.Table S2. Individual ceramide species and risk of incident dementia and AD dementia.Table S3. Individual ceramide species and MRI markers of structural brain injuryClick here for additional data file.


Quantitative skeletal muscle mass loss has the potential to predict the therapeutic effects of immune checkpoint inhibitors. This study aimed to assess the impact of muscular quality on the abovementioned outcomes.2 and ≥25 kg/m2, respectively, as assessed using lumbar computed tomography images. High muscle quantity was stipulated as a lumbar skeletal muscle index ≥41 cm2/m2 in women, ≥43 cm2/m2 in men with a BMI <25 kg/m2, and ≥53 cm2/m2 in men with a BMI ≥25 kg/m2. We evaluated the associations of these muscular parameters with the overall response rate (ORR), progression‐free survival (PFS), and overall survival (OS).This study retrospectively reviewed the medical records of patients with advanced non‐small cell lung cancer (NSCLC) who had received PD‐1/PD‐L1 inhibitor monotherapy between March 2016 and February 2018. High muscle quality was stipulated as a skeletal muscle density ≥41 and ≥33 Hounsfield units in patients with a body mass index (BMI) <25 kg/mp<0.05) and longer PFS durations  than those with low muscle quality. There were no noted differences in the ORR or PFS between patients with high and those with low muscle quantities. On the contrary, regardless of muscle quality and quantity, there were no differences in OS between patients with high and those with low muscle status.Out of 156 patients, 80 (51.3%) and 47 (30.1%) showed low muscle quality and quantity, respectively. Patients with high muscle quality showed higher ORR (35.0 vs. 15.8 %, Lumbar skeletal muscle quality has the potential to predict the therapeutic effect of anti‐programed cell death 1/programed cell death ligand 1 inhibitor monotherapy in patients with advanced NSCLC. High muscle quality correlates with higher objective response rate and longer progression‐free survival duration in treatment with immune checkpoint inhibitors. On the other hand, there is no association between muscle quantity and the efficacy of immune checkpoint inhibitors. The eligibility criteria were as follows: (a) histologically proven stage III or IV NSCLC, including postoperative recurrence, with reference to the seventh or eighth edition of tumor–node–metastasis staging32, or the presence of muscle depletion according to the consensus criteria.The following data were collected from the medical records of eligible patients: age, sex, PS just before the initiation of PD‐1/PD‐L1 inhibitor therapy, histology, stage, PD‐L1 tumor proportion score (TPS) and driver oncogenes when available, number and types of prior treatments, and the type of PD‐1/PD‐L1 inhibitor received. Pretreatment and previously measured body weight values (measured 6 months before the treatment) were collected, and the body mass index (BMI) and weight changes were calculated. Moreover, neutrophil‐to‐lymphocyte ratios (NLRs), which reflect inflammation, were collected with blood test data just before the initiation of PD‐1/PD‐L1 inhibitor therapy. NLR was calculated as neutrophils divided by lymphocytes, and a high NLR was stipulated as an NLR ≥5.3.1The status of ALK translocation and EGFR mutation was assessed in all patients with non‐squamous NSCLC, while the BRAF mutation and ROS‐1 translocation status was assessed in some patients, as appropriate. The PD‐L1 TPS values were calculated by trained pathologists using the 22C3 or 28‐8 pharmDx assay .3.22, respectively (see Table 2/m2) and calculated as [cross‐sectional area of skeletal muscle (cm2)/height2 (m2)].2/m2 in women, ≥43 cm2/m2 in men with a BMI <25 kg/m2, and ≥53 cm2/m2 in men with a BMI ≥25 kg/m2.The cross‐sectional area of the lumbar skeletal muscle at the third level of the lumbar vertebra (L3) was analyzed using electronically stored CT scans that were taken before the initial administration of PD‐1/PD‐L1 inhibitors, with or without contrast enhancement within a 5‐mm slice thickness. Lumbar skeletal muscles were identified based on Hounsfield unit (HU) thresholds of −29 to +150.3.3p‐values were two‐sided, and a p‐value <0.05 was considered statistically significant. All statistical analyses were performed using R.https://cran.r‐project.org/src/base/R‐3/).The primary outcome was PFS, which was defined as the period between the first day of PD‐1/PD‐L1 inhibitor administration and the day of tumor progression or death, whichever occurred first. The final follow‐up was on July 3, 2019. The secondary outcome was overall survival (OS) and ORR. We defined OS as the period between the first day of PD‐1/PD‐L1 inhibitor administration and the day of death from any cause. Median PFS and OS were assessed by the Kaplan–Meier method. Data for patients without disease progression or who were lost to follow‐up were censored at the time of the last CT scan for PFS assessment. For PFS and period of response, data for patients in whom treatment with new anticancer agents were initiated patients in whom treatment with new anticancer agents were initiated without recognizing tumor progression were censored on the day of the final tumor assessment before treatment with new anticancer agents was initiated. Data for patients who were alive or lost to follow‐up were censored for OS when they were last known to be alive. Log‐rank tests were used to compare the PFS and OS curves. According to the Response Evaluation Criteria in Solid Tumors, ORR was measured as the ratio of the sum of complete response and partial response. Potential predictors were assessed using Cox proportional hazards models for PFS. For the crude hazard ratio, the covariates included PD‐L1 expression (TPS ≥50% vs. <50% or unknown), ECOG‐PS (0–1 vs. 2), muscle quality (high vs. low), muscle quantity (high and low), NLR (≥5 vs. <5), and number of prior treatments (1 vs. ≥2 regimens). The hazard ratio for muscle quality was adjusted for PD‐L1 expression and ECOG‐PS. Fisher’s exact test and the Wilcoxon test were performed to compare various characteristics between the low and high muscle quality groups. All 44.1p < 0.05) and mean attenuation . Out of patients with a high muscle quality, 30 (39.5%) and 58 (72.5%) patients had cancer cachexia and a low muscle quantity, respectively.Out of 279 consecutive patients with advanced NSCLC who initially received PD‐1/PD‐L1 inhibitor therapy between March 2016 to February 2018, 156 were finally enrolled in this study . Patients with a high muscle quality had a higher ORR than those with a low muscle quality . Conversely, no difference in the ORR was noted between patients with high and those with low muscle quantity values . We further assessed the response rate for each muscle quality group. There was no difference in the ORR in the high muscle quality group between patients with high and those with low muscle quantity values . Similarly, in the low muscle quality group, no difference in the ORR was noted between patients with high and those with low muscle quantity values .The ORR was 25.6% in all patients . Patients with high tumor PD‐L1 expression (TPS ≥50%) had a higher ORR than those with low or unknown PD‐L1 expression . Patients with high muscle quality had a longer PFS than those with low muscle quality , after adjusting for PD‐L1 expression, pretreatment PS, and number of prior treatments  showed tumor progression up to the cutoff date July 3, 2019). After a median follow‐up of 2.5 months , patients with high tumor PD‐L1 expression (TPS ≥50%) had a longer PFS duration than those with low or unknown PD‐L1 expression  had died before the cutoff date . After a median follow‐up of 11.2  months, no significant difference in OS was noted between patients with high and those with low muscle quality values ‐α, interleukin (IL)‐6, the NLR, and C‐reactive protein in humans.There are some limitations in this study. First, our analysis was limited by its retrospective nature, for which we were unable to exclude biases associated with unknown factors. Second, the small sample size comprises patients from a single institution, limiting our ability to generalize our results to other populations. Third, we did not measure the level of tumor antigenicity by the tumor mutation burden or microsatellite instability because they are not routinely measured in Japanese clinical practice in advanced NSCLC settings. Fourth, there was a possibility that we could not accurately assess the impact of muscle quality on the OS of patients because it did not directly influence the therapeutic effects of PD‐1/PD‐L1 inhibitors in patients with various statuses with regard to PS, treatment line, driver oncogene, and presence of cancer cachexia. Finally, this study did not include a control cohort that did not receive immunotherapy, and therefore, it was difficult to make accurate assessments or conclusions regarding predictive roles. A further prospective study is needed to eliminate these biases and patient heterogeneity for the confirmation of our results.6High lumbar skeletal muscle quality may potentially be used to predict the therapeutic effects of PD‐1/PD‐L1 inhibitors, independent of PS and PD‐L1 expression, in patients with advanced NSCLC. However, further studies are needed to validate our results.T.N. reported grants from Ono Pharmaceutical Co., Ltd., Pfizer US, Inc., and Mochida Pharmaceutical Co., Ltd. that are outside of the submitted work. H.K. reported grants from Eli Lilly K.K and Taiho Pharmaceutical that are outside the submitted work. S.O. reported grants from Chugai Pharmaceutical Co., Ltd., Ono Pharmaceutical, AstraZeneca K.K., Boehringer Ingelheim, Taiho Pharmaceutical, and MSD that are outside the submitted work. K.W. reported grants from Chugai Pharmaceutical Co., Ltd., Taiho Pharmaceutical, Boehringer Ingelheim, Eli Lilly K.K., Ono Pharmaceutical, and MSD that are outside the submitted work. A.O. reported grants from Taiho Pharmaceutical, Ono Pharmaceutical, Chugai Pharmaceutical Co., Ltd., and Novartis Pharma K.K. that are outside the submitted work. H.K. reported grants from Ono Pharmaceutical Co, Ltd., Kyowa Hakko Kirin Co., Ltd., Bristol‐Myers Squibb, MSD, Eli Lilly K.K, Novartis Pharma K.K., Daiichi‐Sankyo Co., Ltd., and Pfizer K.K., and grants and personal fees from AstraZeneca K.K., Chugai Pharmaceutical Co, Ltd., and Boehringer Ingelheim that are outside the submitted work. H.M. reported grants and personal fees from AstraZeneca, Chugai Pharmaceutical Co., Ltd., Taiho Pharmaceutical, and Eli Lilly Japan., and grants from Abbvie, Daiichi Sankyo, and IQvia, and personal fees from Ono Pharmaceutical, Bristol‐Myers Squibb Japan, and MSD that are outside the submitted work. K.T. reported Lecture fee from AstraZeneca, MSD, Chugai‐Roche Co., Ono Pharmaceutical Co., Eli Lilly, Boehringer‐Ingelheim Co., and grants from Chugai‐Roche Co., Ono Pharmaceutical Co. that are outside the submitted work. T.T. reported grants and personal fees from AstraZeneca KK, Pfizer Japan, Inc., Eli Lilly Japan K.K., Chugai Pharmaceutical Co., Ltd., Ono Pharmaceutical Co., Ltd., MSD K.K., and Boehringer Ingelheim Japan, Inc., and personal fees from Roche Diagnostics K.K. that are outside the submitted work. The other authors have no conflict of interest to declare.Naoya Nishioka and Tateaki Naito Conceptualized the research, designed the study, interpreted the results and wrote the manuscript. Akifumi Notsu and Keita Mori oversaw the analyses as biostatisticians. Hiroaki Kodama, Eriko Miyawaki, Taichi Miyawaki, Nobuaki Mamesaya, Haruki Kobayashi, Shota Omori, Kazushige Wakuda, Akira Ono, Hirotsugu Kenmotsu, and Haruyasu Murakami: Collected data. Koichi Takayama and Toshiaki Takahashi: Revised the final version of the manuscript.This retrospective cohort study was approved by the institutional review board of Shizuoka Cancer Center, Japan (J2019‐196), and performed under the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. The need for informed consent was waived owing to the retrospective nature of the study.Table S1Click here for additional data file.


PRKDC) is an important gene for DNA double‐strand break (DSB) repair and central T‐cell tolerance. We aimed to investigate the association between PRKDC mutations and tumor mutation burden (TMB), tumor microenvironment (TME), and response to ICI. Whole‐exome sequencing data of 4023 solid tumor samples from the Cancer Genome Atlas (TCGA) and panel‐based sequencing data of 3877 solid tumor samples from Geneplus‐Beijing, China, were used to analyze the TMB. The mRNA expression data of 3541 solid tumor samples from TCGA were used to explore the effect of PRKDC mutations on the TME. Four ICI‐treated cohorts were analyzed for verifying the correlation between PRKDC mutations and the response to ICI. In both the TCGA and Geneplus datasets, we found that the TMB in PRKDC mutation samples was significantly higher than in PRKDC wild‐type samples . Further, TCGA datasets showed that PRKDC mutation samples were associated with a significantly increased expression of CD8+ T cells, NK cells, immune checkpoint, chemokines, etc. compared to PRKDC wild‐type samples (P < 0.05). In ICI‐treated cohorts, we also found the PRKDC mutations were associated with increased survival , and the increase was significant in multivariate analysis . In summary, we found that PRKDC mutation often appeared to co‐exist with deficiency in some other DNA damage repair mechanism and is nonetheless one of the important factors associated with increased TMB, inflamed TME, and better response to ICI.Predictive biomarkers of response to immune checkpoint inhibitors (ICI) help to identify cancer patients who will benefit from immunotherapy. Protein kinase, DNA‐activated, catalytic subunit ( PRKDC) is a gene related to DNA damage response (DDR). Here, we found that PRKDC mutation often co‐exists with other DNA damage repair deficiencies and is associated with an increased tumor mutation burden, an inflamed tumor microenvironment, and a good response to immune checkpoint inhibitors.Protein kinase, DNA‐activated, catalytic subunit ( DDRDNA damage repairDNA‐PKDNA‐dependent serine/threonine protein kinaseDNA‐PKcsDNA‐dependent serine/threonine protein kinase catalytic subunitDSBDNA double‐strand breakGSEAGene set enrichment analysisHRhomologous recombinationICIsimmune checkpoint inhibitorsMMRmismatch repairMSImicrosatellite instabilityNHEJnonhomologous end‐joiningPRKDCprotein kinase, DNA‐activated, catalytic subunitTCGAThe Cancer Genome AtlasTMBtumor mutation burdenTMEtumor microenvironment1New immune checkpoint inhibitors (ICI) have changed the therapeutic landscape for many types of cancer , 2, 3. HPOLE) and polymerase ƌ (POLD1), mismatch repair (MMR) genes, and BRCA1/BRCA2 reduce genomic stability and can lead to hypermutations [PRKDC) that encodes the DNA‐dependent serine/threonine protein kinase catalytic subunit (DNA‐PKcs) is a member of the DDR pathway. DNA‐dependent serine/threonine protein kinase (DNA‐PK) is composed of DNA‐PKcs and a heterodimer of Ku proteins (Ku70/Ku80). DNA‐PK is a critical component of the nonhomologous end‐joining (NHEJ) pathway that is mainly involved in DNA double‐strand break (DSB) repair and maintaining genomic integrity [PRKDC mutations by reviewing the Cancer Genome Atlas (TCGA) database, the Geneplus database, and four available clinical cohorts treated with ICI [PRKDC mutations were significantly associated with a high TMB similar with other DDR pathway‐related gene mutations, including POLE/D1, MMR genes, BRCA1/2, and the presence of PRKDC mutations predicted a superior response to ICI therapy compared with patients with wild‐type PRKDC. We highlight the importance of validation of PRKDC mutations for the delivery of precise immunotherapy.Gene mutations in the tumor DNA damage repair (DDR) pathway have also been recently reported as an important predictor for the response to ICI therapy. Mutations in DNA polymerase ɛ . DNA from formalin‐fixed, paraffin‐embedded specimens (FFPE) was isolated by using a commercially available kit . The DNA concentration was measured using a Qubit fluorometer and the Qubit dsDNA HS (High Sensitivity) Assay Kit .2.2.2Sequencing was carried out using Illumina 2 × 75‐bp paired‐end reads on an Illumina HiSeq 3000 instrument according to the manufacturer's recommendations using the KAPA DNA Library Preparation Kit . Barcoded libraries were hybridized to a customized panel of 1021 genes containing whole exons and selected introns of 288 genes and selected regions of 733 genes, and another panel of 430 genes most frequently mutated in solid tumors. The libraries were sequenced to a uniform median depth (> 500×) and assessed for somatic variants including single nucleotide variants (SNVs), small insertions and deletions (InDels), copy number alterations (CNA), and gene fusions/rearrangements.2.2.3MuTect2 (3.4‐46‐gbc02625)  was empl2.32.3.12(x + 1)‐transformed before analysis.We obtained the WES data of 4023 solid tumors and the mRNA expression data of 3541 solid tumors across 10 tumor types from TCGA. The experimental procedures for DNA and RNA extraction from tumors, library preparation, sequencing, quality control, and subsequent data processing were published previously by TCGA . The mRN2.3.2Gastric, colorectal, and endometrial tumors are the three types of cancer known to include microsatellite stability (MSS) and microsatellite instability (MSI) subtypes. To explore the association between PRKDC mutations and MSI status and their effects on TMB, we obtained the WES data from 246 endometrial carcinoma, 223 colorectal adenocarcinoma, and 295 gastric adenocarcinoma samples with MSS/MSI subtype information from three studies , 21, 22 2.3.3PRKDC mutations and the clinical benefits of ICI, we analyzed the genomic and clinical data from four clinical cohorts treated with ICI and predicted the neoantigen data from the Hellmann cohort. The first cohort consisted of 75 patients with non‐small‐cell lung cancer (NSCLC) treated with anti‐PD‐1 therapy and anti‐CTLA‐4 therapy (Hellmann cohort) [To further explore the association between  cohort) . The sec cohort) . The thi cohort) . The las2.4We defined any nonsynonymous mutations in the gene of interest as ‘mut+’, including missense, nonsense, frameshift indels, in‐frame indels, and splice site mutations.2.4.1Tumor mutation burden was defined as the total somatic nonsynonymous mutation counts in coding regions. TMB was classified into high or low taking the top quartile as the cutoff value.2.4.2reg, CD4 T cells, neutrophils, etc. The mRNA expression of a gene set was defined as the arithmetic mean of transcripts per million values of genes in this gene set.The response to ICI has been reported to be related to cytotoxic T cells, NK cells, chemokines, and checkpoints , 24. The2.4.3javagsea 3.0 Desktop Application (http://software.broadinstitute.org/gsea/index.jsp). The gene sets used for the enrichment analysis were downloaded from the Molecular Signatures Database . The gene sets with a false discovery rate (FDR) < 0.05 were considered as significantly enriched. The normalized enrichment score (NES) is the primary statistic for examining gene set enrichment results.Gene set enrichment analysis (GSEA) was performed using the 2.4.4https://github.com/hammerlab/topiary/) [netmhccons (v. 1.1) [50 of % 500 nm to one (or more) of the patient‐specific HLA alleles.Neoantigens in 75 samples from the Hellmann cohort were estimated . The mutopiary/) . Topiary(v. 1.1)  in order2.5PRKDC mutations on protein function in a clinical case we presented. Mutations with a SIFT score < 0.05 predicted to be deleterious or PolyPhen‐2 HumVar score > 0.5–0.9 were considered possibly damaging and probably damaging (score > 0.9).We used the functional impact predicting tools SIFT and PolyPhen‐2 HumVar to predict the effects of 2.6graphpad prism  and spss version 25.0 , Armonk, NY, USA). If TMB, TNB, and mRNA were normally distributed, a Student t test was used to determine the differences between two groups; otherwise, the Mann–Whitney U test was used. Logistic regression was used to analyze the influencing factors of TMB‐high. Pearson's correlation was used to analyze the correlation between the length of exons and the mean number of somatic mutations in the exon region for 800 genes with long transcripts including PRKDC, and the fitness lines in scatter plots were plotted with Loess regression which fitted by (weighted) least squares. Kaplan–Meier survival and multivariate Cox regression analyses were used to analyze associations between mutation type and survival, with a P value determined by a log‐rank test. HR was determined through Cox regression. The factors associated with survival with P < 0.15 in univariable analysis were included in Cox proportion hazard model multivariable analysis. All reported P values were two‐tailed, and P < 0.05 was considered significant.Statistical analyses were conducted using 33.1PRKDC mutations in the TCGA cohort and in the Geneplus cohort (a cohort of a Chinese pan‐cancer population). In the TCGA cohort, colorectal adenocarcinoma had the highest PRKDC mutation frequency of 9.66% (51/528), followed by gastric adenocarcinoma with 9.63% (42/436), endometrial cancer 9.27% (23/248), and non‐small‐cell lung cancer 7.86% . Other results are shown in , POLE/POLD1, and BRCA1/BRCA2 [P < 0.05; Table To validate the association of  in Fig. . PreviouA1/BRCA2 . Our stuPRKDC mutations showed significantly higher TMB than those with PRKDC wild‐type . When compared with other DDR genes, the PRKDC mutation group had a higher TMB than the BRCA1/2 mutation group (P < 0.05) but was not different from the MMR mutation group or the POLE/D1 mutation group. The proportion of TMB‐high in the PRKDC mutation group was 74% over a pan‐cancer analysis  of patients with other DDR genes  mutation, but four of the rest six PRKDC mut+/MSS MSI‐L/other DDR mut− patients were still TMB‐H. Then, we compared the TMB among PRKDC mut+/MSI‐H, PRKDC mut−/MSI‐H, PRKDC mut+/MSS MSI‐L, and PRKDC mut−/MSS MSI‐L subgroups in the four cohorts. In the Lander's endometrial carcinoma cohort [PRKDC mut+ samples is significantly higher than in the PRKDC mut− samples in both MSI‐H and MSI‐L/MSS subtypes . In the Muzny's colorectal adenocarcinoma cohort [PRKDC mut+ samples is significantly higher than in the PRKDC mut− samples only in the MSS/MSS‐L subtypes . In the Kai Wang's gastric adenocarcinoma cohort [PRKDC mut+ samples is significantly higher than in the PRKDC mut− samples only in the MSI‐H subtypes . In most of the genes, we found that the length of the transcript correlated with the average number of somatic mutations within them when we analyzed four TCGA patient cohorts  in the PRKDC mutation group  was superior to that of PRKDC wild‐type patients [median, not reached (NR) vs. 6.8 months; HR, 0.2893; 95% CI, 0.1255–0.6672; P = 0.0650, Fig. PRKDC mutation and those with a PRKDC wild‐type were 66.67% and 32.26%, respectively.The Hellmann cohort included 75 patients with NSCLC, including six patients with PRKDC mutations. Compared with the patients with the PRKDC wild‐type, the PRKDC mutation patients in this cohort also trended toward a longer overall survival  in the two cohorts. In the Hellmann cohort, we found that PRKDC mutations were significantly associated with a longer PFS than in the none mutation subgroup . The PD‐L1 expression by IHC staining in both tumor cells and immune cells was positive  with stage IVA nasopharyngeal carcinoma that harbored a ons Fig. . The ctDons Fig. . When dions Fig. .4PRKDC mutations in two large cohorts was identified in multiple solid tumor types, including common neoplasms such as gastrointestinal cancers, NSCLC, and bladder carcinoma. We are the first to comprehensively describe that PRKDC mutations, regardless of the status of other DDR‐related genes, and found an association with an increased TMB, an increased mRNA expression of immune‐related genes, and a superior response to ICI in pan‐cancer patients.In our study, the prevalence of PRKDC mutations lead to a deficiency in the DNA‐PKcs and NHEJ pathway, so DSB fail to repair and mutations tend to accumulate. Furthermore, DNA‐PKcs has been verified as essential for induction of apoptosis after massive DSB formation [PRKDC mutations are present, cancer cells are resistant to apoptosis and there is an increase in the accumulation of DNA damage that promotes genome instability. Moreover, such excess DNA damage may not only increase mutations due to error‐prone translesion synthesis, but also increase epigenetic alterations due to errors during DNA repair [PRKDC mutations and found they were significantly associated with high TMB scores in two large independent cohorts, similar to POLE/POLD1, MMR gene, and BRCA1/BRCA2 mutation patients. This finding implicates PRKDC mutations as a valuable biomarker in clinical practice.Double‐stranded DNA breaks are the most serious DNA lesions. The two major pathways for repair of DSB are homologous recombination (HR) and NHEJ , 32. DNAormation . When PRA repair , 35. In PRKDC wild‐type, the patients with PRKDC mutations tend to have an inflamed tumor microenvironment (TME) that includes higher numbers of CD8+ T cells, NK cells, Th1, and pDCs, and higher PD‐L1 expression, other immune checkpoints, and chemokine expression. The GSEA analysis also showed a remarkably upregulated expression of the IFN‐γ and IFN‐α response, and IL‐6/JAK/STAT signaling along with downregulation of TGF‐β and Wnt/β‐catenin signaling. The high TMB might be one of the reasons contributing to this inflammatory microenvironment. A previous study has shown that DNA‐PK also interacts with the transcription factor autoimmune regulator (AIRE) to promote central T‐cell tolerance [PRKDC mutations induced inflamed TME was caused by an increased TMB level together with impaired central immune tolerance. In the four independent cohorts, we also found that patients with PRKDC mutations were more likely to benefit from ICI.When performing transcriptome analysis we found that, compared to the olerance . Deficieolerance . These rPRKDC mutation‐induced changes in the immune microenvironment and improvements of ICI treatment efficacy needs to be confirmed and verified in a larger population with multiple cancer types, such as MSS colorectal cancer and pancreatic cancer.However, our study has limitations. First, due to the relatively low incidence and limited data, we can only conclude that PRKDC mutation is one of the important factors which affect TMB, TME, and the prognosis of ICI therapy, but cannot confirm the extent to which PRKDC mutation sites contribute to the inflamed TME and prognosis improvements. Second, the sample size of the validation cohort was small and only melanoma and NSCLC were included, and so, the association between 5PRKDC mutations occur in a subset of solid tumor patients, and they often appeared to co‐exist with deficiency in some other DDR mechanism. But anyway, PRKDC mutation is still one of the important factors significantly associated with an increased TMB, an increased expression of immune‐related genes, and an improved response to ICI. Additional prospective studies are needed to validate this finding and to determine whether routine testing for this alteration is warranted. This work may have important implications for clinical practice and provide a potential predictive biomarker for guiding ICI therapy.Our findings suggest that The authors declare no conflict of interest.YC, CC, and YL conceived and designed the study. YG, JL, GC, LC, and XY made the collection of clinical data and sample. YG, JL, LC, and XY performed sequencing. YH, JL, LC, LP, ZG, NX, and JP analyzed the clinical data and performed statistical analysis. All authors wrote the manuscript. All authors read and approved the final manuscript.Fig. S1. Venn diagram of the relationship among PRKDC mutation subgroup, MSI‐H subgroup and TMB‐H subgroup. .Fig. S2. Comparison of TMB in different combinations of PRKDC mutations and MSI status groups. .Fig. S3. Length of transcript versus the mean number of somatic mutations in coding region for each gene in the TCGA cohorts of Bladder cancer, Colorectal cancer, Lung Adenocarcinoma and Head/Neck Squamous cell carcinoma. R, Pearson's correlation; curve, fitness lines. The statistics used in this analysis is Loess regression.Fig. S4. The hematoxylin‐eosin (HE) staining and immunohistochemistry (IHC) staining of CD8 and PD‐L1 with the recurrent nasopharyngeal lesion of patient NPC_Y. The representative microscopic findings are shown.Fig. S5. Disease course and clinical response in an advanced nasopharyngeal carcinoma patient (NPC_Y) with PRKDC mutation treated with Nivolumab‐based multi‐combination strategies.Table S1. The cancer types and number of samples included in Geneplus pan‐cancer cohort.Table S2. The logistic regression of factors influencing TMB‐high in TCGA top 10 cancers.Table S3. The logistic regression of factors influencing TMB‐high in the combined four cohorts which with MSI/MSS data.Table S4. GSEA between PRKDC mutation group and non‐mutation group with the Hallmark gene set in TCGA top 10 cancers dataset.Table S5. Gene list of immune‐related gene set.Table S6. Immune‐related gene set mRNA expression analysis.Table S7. The clinical data of three patients with PRKDC mutation from two clinical cohorts treated with ICIs.Table S8. The mutation list of an advanced nasopharyngeal carcinoma patient (NPC_Y) by ctDNA analysis.Appendix S1. Case report.Appendix S2. Immunohistochemical staining and analysis for CD8, PD‐L1.Click here for additional data file.


Neonatal acidaemia at birth can increase neonatal morbidity and mortality and it is predictive of neonatal asphyxia. The umbilical blood gas analysis is a valid tool for the evaluation of neonatal acidaemia. However, umbilical cord blood gas analysis is commonly performed in high-risk situations or in the setting of Apgar scores < 7 at 5 min.A retrospective cohort study was conducted from June to December 2018 at the Department of mother’s and child’s health, Poliambulanza Foundation Hospital Institute. Inclusion criteria were: full term newborns with body weight appropriate for gestational age, born by vaginal delivery or caesarean section, reassuring Apgar Score > 7 at 5 min, arterial cord blood gas analysis showing pH < 7.4 or BE <-8 mmol/l or lactate > 6 mmol/l. The aim was to evaluate the predictive role of blood gas analysis for respiratory distress syndrome in newborns with reassuring Apgar Score.2(1) = 10,084, OR (95% CI): 3,9 × 10− 4; p < 0,05). ROC curve revealed that the cut-off point of pH was 7.12, with a sensibility and specificity of 68 and 63%, respectively.352 full term newborns were enrolled. Umbilical cord blood artery pH showed an association with respiratory distress syndrome (χUmbilical cord blood artery pH < 7.12 at birth is associated to respiratory distress syndrome in newborns. Blood gas analysis is an important instrument to help health care providers during assistance in the delivery room, but also to early identify newborns at high risk for respiratory distress syndrome and better manage the care of these newborns after birth. V and in newborns with a high risk of asphyxia. Nowadays, there are not clear threshold value of pH, BE and lactate. It is not clear if newborns with moderate acidaemia and Apgar score ≥ 7V must be monitored for development of adverse outcome. Newborns with a good Apgar score have a residual risk of neonatal acidaemia and adverse outcomes [At birth, each newborn is given a score calculated using the Apgar score. This system is a rapid method to assess the clinical status of the newborn at 1 min and 5 min after birth. The score was formulated to have a rapid assessment of the clinical status of the newborn. Therefore, the Apgar score offers the opportunity to report the status of the newborn after birth and the response to resuscitation when performed. The Apgar index is a score obtained from the evaluation of 5 parameters  for each parameter a score is assigned by the operator . For thioutcomes , 9. Our A retrospective cohort study was conducted from June to December 2018 at the Department of mother’s and child’s health, Poliambulanza Foundation Hospital Institute. The study was conducted in accordance with the Declaration of Helsinki. In this study were enrolled full term newborns with body weight appropriate for gestational age, born by vaginal delivery or instrumental labor or by caesarean section and that showed at blood gas analysis by arterial umbilical cord the following parameters: pH < 7.4, BE <-8 mmol/l, lactate > 6 mmol/l. Preterm newborns were excluded from the study. In delivery room physicians or midwives evaluated Apgar Score at 1 min and 5 min and collected arterial umbilical cord blood within 15 min from delivery. BGA evaluation was a routine practice at birth. Neonatologists were present at all instrumental deliveries and caesarean sections but only at the request of midwives during vaginal deliveries All babies received the same clinical management the environment was strictly controlled, the delivery room temperature did not have to rise less 26 °C , while tThe following clinical features of the newborns were recorded in a database: gender, gestational age, body weight, Apgar score, pH, BE, lactate, delivery mode, hypoglycaemia at 2 h, hypothermia, admission in NICU, RDS. Nurses that were on duty measured the body weight of the newborn by Eura Mod.AS/1 O.M.I.P. Milano and temperature by Filac™ 3000 AD electronic thermometer. Glycaemia and BGA were measured by ABL90 FLEX (Radiometer). RDS is recognised as any signs of breathing difficulties in the neonate (Silverman score) by trained physicians , 6.p < 0,05.A statistician that was aware of the study aims using IBM SPSS Statistics for Windows, v.25, carried out statistical analysis . Sample size was computed setting α = 0.05, β = 0.05, Odds Ratio = 4.6  and obtap > 0,05). Newborns born by these two modalities of birth presented higher pH values than those born by urgency/emergency caesarean section or instrumental labor . Similar pH values were revealed in newborns born by instrumental labor or emergency/urgency caesarean section . Analysing BGA parameters, pH showed an association with RDS . The cut-off point of pH was 7.10 with sensibility, specificity, PPV and NPV of 68, 78, 29 and 95% respectively, while the cut-off point of lactate was 8.25 mmol/l with sensibility, specificity, PPV and NPV of 68, 81, 30 and 94% respectively  =4749; p < 0,05; OR = 0,995 . The overall incidence of umbilical artery pH < 7.12 was 33.8% (N = 119). Out of these 119 newborns, 14% showed RDS, 24% had access in NICU and 15% received neonatal resuscitation.A total of 352 full term newborns were enrolled for the study. Clinical characteristics of population study were reported in Table DS Table , with a p = 0.14, odds = 0.08, positive likelihood ratio = 0,06)RDS given a pH < 7.12 Access in NICU given a pH < 7.12 Neonatal resuscitation given a pH < 7.12 (The probability that one of the following conditions occurred in newborns were:Acidaemia and RDS occurred in 4.7% of the enrolled newborns, while acidaemia and access in NICU occurred in 8% of enrolled newborns.V should be monitored for the development of adverse neurological outcome. Sabol et all reported that newborns with a good Apgar the score, have a risk of neonatal acidaemia and adverse outcome [2 and heart rate by pulse oximeter, respiratory rate, sucking, colour skin, temperature and the appearance of any breathing difficulties.Our study was conducted to evaluate the predictive role of pH, BE and lactate in umbilical cord BGA, for respiratory outcome of newborns after birth. Umbilical cord blood gas analysis is important to evaluate the newborn acidaemia during childbirth . Actuall outcome . Moreove outcome . Our res outcome . pH is aArterial umbilical cord BGA seems to be a useful tool to early identify newborns at high risk to develop RDS. Arterial cord blood pH < 7.12 even in presence of reassuring Apgar Score represents a risk factor for RDS onset. Hypothermia and hypoglycaemia at birth are additional risk factors for RDS onset. Furthermore, pH, BE, lactate and hypothermia are predictive for the access in NICU.


To identify lesional and nonlesional tissues from early gastric cancer (EGC) patients by Raman spectroscopy to build a diagnostic model and effectively diagnose EGC.  Specimens were collected by endoscopic submucosal dissection from 13 patients with EGC, and 55 sets of standard Raman spectral data (each integrated 10 times) were obtained using the fiber optic Raman system; there were 33 sets of lesional tissue data, including 18 sets of high-grade intraepithelial neoplasia (HGIN) data and 15 sets of adenocarcinoma data, and 22 sets of nonlesional tissue data. After the preprocessing steps, the average Raman spectrum was obtained. −1, 1103 cm−1, 1417 cm−1, 1206 cm−1, 1234 cm−1, 1479 cm−1, 1560 cm−1, and 1678 cm−1. Compared with the peaks corresponding to nonlesional tissues, the peaks of the lesional tissues shifted by different magnitudes, and a new characteristic peak at 1324 cm−1 was observed. Comparing the peak intensity ratio and the integral energy ratio of the lesional tissues with those of the nonlesional tissues revealed a significant difference between the two groups . Considering the peak intensity ratio of I1560 cm−1/I1103 cm−1 as a diagnostic indicator, the accuracy, sensitivity, and specificity of diagnosing EGC were 98.8%, 93.9%, and 91.9%, respectively. Considering the integral energy ratio (noncontinuous frequency band and continuous frequency band) as a diagnostic indicator, the accuracy, sensitivity, and specificity of diagnosing EGC were 99.2-99.6%, 93.9-97.0%, and 95.5%, respectively.  The nonlesional tissues showed peaks at 891 cm The integral energy ratio of the Raman spectrum could be considered an effective indicator for the diagnosis of EGC. Gastric cancer is one of the most common malignancies in the world, and its incidence and mortality rates continue to be high, making it a serious threat to human health. According to the latest statistics, there were approximately 1,033,000 new gastric cancer cases and 783,000 related deaths in the world in 2018, ranking gastric cancer sixth in incidence and third in mortality among malignancies . TherefoCurrently, the methods for diagnosing gastric cancer mainly include serology, imaging, and endoscopic biopsy. Among these methods, the specificity and sensitivity of serological tests are poor, and increases in indexes mostly appear in advanced gastric cancer, causing this approach to be used more often as an auxiliary indicator for diagnosing advanced gastric cancer and monitoring for recurrence and progression after gastric cancer surgery in clinical work . ImagingRaman spectroscopy is an inelastic scattering technique that is performed by shifting the frequency of material molecular vibrations with incident light. The obtained Raman spectra exhibit different characteristics as the molecular content and composition of the material change. The structure, content, and conformation of various components in cells will change to different extents during the process of carcinogenesis; thus, Raman spectroscopy could be used to identify differences between the spectra obtained from early cancer tissues and those obtained from healthy tissues , 7. TherRaman spectroscopy has been widely used in the diagnosis of various tumors, such as skin tumors and oral tumors and tumors of gastric cancer, esophageal cancer, ovarian cancer, and prostate cancer –13. Mostμm in diameter, N.A. = 0.22) surrounding the central excitation fiber , with two filters incorporated at the proximal and distal ends of the probe to maximize the collection of tissue Raman signals while reducing interference from light other than Raman-scattered light. (3) A Raman imaging spectrometer . (4) A charge-coupled device (CCD) camera . (5) A monitor . (6) Andor SOLIS software. The fiber optic Raman spectroscopy system is shown in (1) A near-infrared semiconductor laser with an excitation wavelength of 785 nm . (2) A fiber optic Raman probe : the Raman fiber optic bundle was 3 m in length and had an outer nylon protective sleeve composed of 7 collection fibers  and 7 cases of adenocarcinoma. All patients in the study volunteered and signed informed consent forms permitting the investigative collection of ESD specimens and Raman spectral data. The study was performed in accordance with the human trial ethics standards and was approved by the hospital ethics committee.−1, the exposure time was set as 1 second, the number of integrations was set as 10, the CCD temperature was set as -80°C, and the electrical current was set as 66 mA. The specimen was placed on an aluminized mirror, and then, the most severely affected area of the lesional tissue was selected after observation by ME. Next, 2-5 points were selected depending on the size of the most severely affected area. After the measurement was completed, the site of the measured point was marked accurately. Then, several sites of nonlesional tissue around the lesional tissue were selected and measured by the same method. After each measurement was completed, the site of the measured point was marked accurately. The specimen was fixed, soaked in 10% formalin solution, and sent to a histopathologist for double-blind histopathological examination. According to the histopathological results, we restored the shape of the ESD specimen, determined the area confirmed as EGC and excluded data that did not meet the criteria . A total of 55 sets of Raman spectral data were collected, including 15 sets of HGIN Raman spectral data, 18 sets of adenocarcinoma Raman spectral data, and 22 cases of nonlesional Raman spectral data. ESD specimens were immediately sent to the Raman spectrum collection room without any processing after using ME to determine the most severely affected area of the lesional tissue. Then, the Raman spectral range was set as 0-2000 cmDue to the background autofluorescence of tissue and external disturbances, the original measured Raman spectral data were at a high baseline level, and the Raman spectral signal was weak. The data format was converted by Andor SOLIS, the baseline was lowered, and the strong background fluorescence was removed by Origin Pro 8.0 software. Next, the Raman signal was highlighted, the curve was smoothed by a fast Fourier transform (FFT), and the baseline level was adjusted. Then, we obtained smooth Raman spectral data with a high signal-to-noise ratio Figures  and 6.t-tests were used to compare the integrated energy ratio and peak intensity ratio between the EGC and nonlesional tissue samples using SPSS 19 software. Then, the receiver operating characteristic (ROC) curve was drawn, and the area under the curve (AUC) was calculated.Independent-samples −1 (tyrosine), 852 cm−1 , 891 cm−1 (saccharide), 992 cm−1 (unknown), 1103 cm−1 , 1171 cm−1 , 1206 cm−1 (tyrosine), 1234 cm−1 (amide III), 1292 cm−1 (cytosine), 1417 cm−1 (C=C stretching in quinoid ring), 1479 cm−1 (amide II), 1517 cm−1 (β-carotene accumulation), 1560 cm−1 (tryptophan), 1634 cm−1 (amide I), 1678 cm−1 (NADH), and 1729 cm−1 (ester group). In contrast, significant Raman peaks corresponding to the lesional tissues, including HGIN and adenocarcinoma, were observed in the Raman spectra at 826 cm−1, 888 cm−1, 977 cm−1, 987 cm−1, 1101 cm−1, 1171 cm−1, 1209 cm−1, 1236 cm−1, 1294 cm−1, 1324 cm−1 (collagen and purine bases of DNA), 1413 cm−1, 1517 cm−1, 1562 cm−1, and 1682 cm−1. −1, 838 cm−1 (amine deformation vibrations), 849 cm−1, 861 cm−1, 875 cm−1 (tryptophan), 889 cm−1, 987 cm−1, 1101 cm−1, 1171 cm−1, 1209 cm− 1, 1235 cm−1, 1253 cm−1 (C-O4 aromatic stretching), 1324 cm−1, 1413 cm−1, and 1562 cm−1, while the Raman spectrum of the adenocarcinoma tissues displays significant peaks at 820 cm−1 , 849 cm−1, 861 cm−1, 875 cm−1, 887 cm−1, 948 cm−1 , 977 cm−1, 1102 cm−1, 1171 cm−1, 1209 cm−1, 1236 cm−1, 1293 cm−1, 1323 cm−1, 1413 cm−1, 1517 cm−1, 1531 cm−1 , and 1562 cm−1. The assignments for the peaks are given in The average Raman spectra of the lesional and surrounding nonlesional tissues in EGC ESD specimens are shown in −1, the peak intensity of the nonlesional tissues was obviously higher than that of the lesional tissues, while the peak intensity in the range of 1350-1800 cm−1 displayed the opposite trend. Compared with those of the nonlesional tissues, the Raman peaks of the lesional tissues at 826 cm−1, 1171 cm−1, and 1517 cm−1 remained fixed. In contrast, the Raman shifts of the spectral peaks of the lesional tissues at 888 cm−1, 1101 cm−1, and 1413 cm−1 moved toward lower wavenumbers than those of the nonlesional tissues at 891 cm−1 (saccharide), 1103 cm−1 , and 1417 cm−1 (C=C stretching in quinoid ring), while the Raman shifts of the spectral peaks of the lesional tissues at 1209 cm−1, 1236 cm−1, 1294 cm−1, 1480 cm−1, 1562 cm−1, and 1682 cm−1 moved toward higher wavenumbers than those of the nonlesional tissues at 1206 cm−1 (tyrosine), 1234 cm−1 (amide III), 1292 cm−1 (cytosine), 1479 cm−1 (amide II), 1560 cm−1 (tryptophan), and 1678 cm−1 (NADH). Additionally, a characteristic peak was observed at 1324 cm−1 in the average Raman spectrum of the lesional tissues, which can be assigned to the purine bases in DNA.(1) There were obvious differences between the average Raman spectra of the lesional and nonlesional tissues in the EGC ESD specimens, as shown in −1, 861 cm−1 (tyrosine), 875 cm−1 (tryptophan), 1171 cm−1, 1209 cm−1, 1413 cm−1, and 1562 cm−1 were the same. Furthermore, compared with those of the adenocarcinoma tissues at 887 cm−1, 1102 cm−1, 1236 cm−1, and 1323 cm−1, the Raman shifts of the spectral peaks of the HGIN tissues at 889 cm−1, 1101 cm−1, 1235 cm−1, and 1324 cm−1 were only slightly different. In the average Raman spectrum of the HGIN tissues, peaks were clearly observed at 826 cm−1, 838 cm−1 (amine deformation vibrations), and 1253 cm−1 (C-O4 aromatic stretching), while no related peaks were observed in the adenocarcinoma tissue spectrum. Additionally, peaks were observed at 820 cm−1 , 948 cm−1 , 1293 cm−1, 1517 cm−1, and 1531 cm−1  in the adenocarcinoma tissues, while no corresponding peaks were observed at 820 cm−1, 948 cm−1, and 1293 cm−1 in the HGIN tissues, and the related peaks at 1517 cm−1 and 1531 cm−1 were not obvious.The average Raman spectrum of the HGIN tissues was very close to that of the adenocarcinoma tissues in the lesional area of the EGC ESD specimens, as shown in −1/E1050-1150 cm−1) and the continuous frequency band (E1350-1500 cm−1/E1200-1350 cm−1) were selected. The two sets of integral energy ratio data acquired from the lesional and nonlesional tissues were analyzed by independent-samples t-test, revealing a significant difference between the two sets of data (P < 0.01). The ROC curve was drawn by the SPSS 19 software. The AUC of the integral energy ratio in the noncontinuous frequency band (E1500-1600 cm−1/E1050-1150 cm−1) was 0.992, meaning that with this noncontinuous frequency band, the accuracy of identifying EGC was 99.2%, and the corresponding sensitivity and specificity were 93.9% and 95.5%, respectively. The AUC of the integral energy ratio in the continuous frequency band (E1350-1500 cm−1/E1200-1350 cm−1) was 0.996, meaning that with this continuous frequency band, the accuracy of identifying EGC was 99.6%, and the corresponding sensitivity and specificity were 97.0% and 95.5%, respectively.(2) The Raman spectra of the lesional and nonlesional tissues were analyzed by the integral energy ratio. The noncontinuous frequency band (E1500-1600 cm−1/E1050-1150 cm−1) and the continuous frequency band (E1350-1500 cm−1/E1200-1350 cm−1) were selected. The two sets of integral energy ratio data acquired from the HGIN and adenocarcinoma tissues were also analyzed by independent-samples t-test, revealing no significant difference between the two sets of data (P > 0.05).Then, the Raman spectra of the HGIN and adenocarcinoma tissues were also analyzed based on the integral energy ratio. The noncontinuous frequency band . The ROC curve was drawn by SPSS 19 software. The AUC of the peak intensity ratio of I1560 cm−1/I1103 cm−1 was 0.988, meaning that with this peak intensity ratio, the accuracy of identifying EGC was 98.8%, and the corresponding sensitivity and specificity were 93.9% and 91.9%, respectively.(3) The Raman spectra of the lesional and nonlesional tissues were analyzed by the traditional peak intensity ratio, and the peak intensity ratio of I1560 cm−1/I1103 cm−1 was selected. The two sets of peak intensity ratio data acquired from the HGIN and adenocarcinoma tissues were also analyzed by independent-samples t-test, revealing no significant difference between the two sets of data (P > 0.05).Then, the Raman spectra of the HGIN and adenocarcinoma tissues were also analyzed by the traditional peak intensity ratio, and the peak intensity ratio of I1560 cmThe integral energy ratio and peak intensity ratio data are shown in The Raman effect was first discovered by the Indian scientist CV Raman and published in Nature on March 31, 1928 . The Ram−1/I1103 cm−1 was significantly higher in the EGC tissues than in the nonlesional tissues, as demonstrated by independent-samples t-test. The average value in the nonlesional tissue group was 0.30, which is lower than that in the EGC tissue group (0.68). The P value was 0.000 < 0.01, showing a significant difference, and the accuracy, sensitivity, and specificity indicated in the ROC curve were 98.8%, 93.9%, and 91.9%, respectively.Studies on patients with gastric cancer or esophageal cancer have shown that the levels of phenylalanine in the serum, urine, and stomach contents of patients with tumors are higher than those in healthy people –28. The −1 was assigned to tryptophan, and the peak at 1103 cm−1 was assigned to phenylalanine, indicating that the tryptophan level in EGC tissues was greater than that in nonlesional tissues. Accordingly, tryptophan may serve as a potential tumor marker, providing a basis for the diagnosis of EGC, but further study is still necessary.The peak at 1560 cm−1 (saccharide), 1103 cm−1 , and 1417 cm−1 (C=C stretching in quinoid ring) moved to lower wavenumbers than those of nonlesional tissues, while the other peaks at 1206 cm−1 (tyrosine), 1234 cm−1 (amide III), 1294 cm−1 (cytosine), 1479 cm−1 (amide II), 1560 cm−1 (tryptophan), and 1678 cm−1 (NADH) moved to higher wavenumbers. This upward shift indicates an increase in the energy of vibration. It is believed that changes in the structure of tumor tissue may occur and that proteins, nucleic acids, and other compounds may become more stable because of the introduction of a new ligand or the activation of a surrounding ligand. This result confirms that compared with nonlesional tissues, tumor tissues show obvious changes in terms of the structure of proteins, saccharides, and nucleic acids. A characteristic peak was observed at 1324 cm−1 in the lesional tissues, which was assigned to collagen and the purine bases in DNA, but it was not observed in the nonlesional tissues, indicating that it may be related to changes in the DNA structure in tumor tissues and active nuclear division.The Raman peaks of the lesional tissues at 891 cm−1 in the nonlesional tissues. A peak could also be observed at 987 cm−1 in the HGIN tissues and at 977 cm−1 in the adenocarcinoma tissues, showing a trend toward lower wavenumbers. There have been no relevant reports in the literature regarding the substance to which the characteristic peak may be assigned. The other characteristic peaks were clearly characterized, and there was good correspondence between the peaks of the lesional and nonlesional tissues. Therefore, it was assumed that there was one kind of molecule with a peak located near 992 cm−1 and that the peak shifted from 992 cm−1 in the lesional tissues because of the effects of HGIN and adenocarcinoma. However, this assumption has not yet been confirmed. Another assumption was related to the use of ESD biopsy specimens in this experiment, while most studies reported to date have used biopsy specimens obtained by endoscopy. The histopathological results clearly indicated negative peripheral margins, meaning that the surrounding tissue was nonlesional tissue; however, the surrounding tissue may have been affected by the diseased tissue in the center, resulting in atrophy or intestinalization and causing certain changes at the molecular level because of the proximity to the diseased tissue, which may have caused the appearance of the characteristic peak at 992 cm−1. The above two assumptions still need to be confirmed by further experiments, and the next research step could be exploring differences in Raman spectra between normal and nonlesional tissues.A significant peak was observed at 992 cm−1 was high, but the sensitivity and specificity were lower than those of the integral capacity ratio. Additionally, the accuracy, sensitivity, and specificity of the integral energy ratio in the noncontinuous frequency band and continuous frequency band were all better than those of the traditional mode peak intensity ratio . Moreover, using the peak intensity ratio of a single peak increases the occurrence of errors, while calculating the integral energy ratio within a certain area reduces the occurrence of errors. Therefore, the integral energy ratio of the Raman spectrum could be considered an effective indicator for the diagnosis of EGC.In this experiment, the integral energy ratio and the peak intensity ratio were used as indicators to diagnose EGC. The results show that the accuracy of the peak intensity ratio of I1560 cm-1/I1103 cmCurrently, the combination of ME-NBI and traditional white-light endoscopy is usually used to diagnose EGC in the clinic. When suspicious lesions are found under white-light endoscopy, observation under ME-NBI is often recommended in addition to biopsy. A meta-analysis in 2015 , includi−1) in their Raman shift, and certain peaks were different from their distribution, which proves that there are certain differences between HGIN and adenocarcinoma tissues on Raman spectroscopy. The peak intensities in the two sets of data were also similar. However, the integral energy ratio and traditional peak intensity ratio were also used to analyze the differences between the HGIN and adenocarcinoma tissues, and no significant differences between the two groups were found, which means that there were no significant differences between the Raman spectra of the two pathological types. Considering that a small sample size may not fully reflect the differences in the Raman spectra between the two groups, expanding the sample size is required to further explore the differences between the two groups.The integral shape and peak distribution of the Raman spectra of the HGIN and adenocarcinoma tissues were very similar, and approximately 50% percent of all peaks were completely consistent. Among the other peaks, some showed only slight changes (1-2 cmThis study explored only the differences between HGIN and adenocarcinoma tissues. Answering the questions of whether the Raman spectra of EGC tissues of different pathological types are different and whether the Raman spectra of EGC and advanced gastric cancer tissues are different requires further study. This study preliminarily proves that it is feasible to identify the differences between HGIN and adenocarcinoma tissues and peripheral nonlesional tissues in ESD specimens by Raman spectroscopy, which provides a research basis for defining lesion boundaries by Raman spectroscopy. However, large amounts of sample data still need to be collected for analysis. In the next research step, we will collect more sample data, reduce the error, combine in vivo detection with in vitro detection, establish an initial diagnostic database of Raman spectra, and establish a standard for the diagnosis of EGC by Raman spectroscopy.