AT‐rich interaction domain 1A (ARID1A) is an essential subunit of the switch/sucrose non‐fermentable chromatin remodeling complex and is considered to be a tumor suppressor. The Cancer Genome Atlas (TCGA) molecular classification has deepened our understanding of gastric cancer at the molecular level. This study explored the significance of ARID1A expression in TCGA subtypes of gastric adenocarcinoma.ARID1A, and obtained correlations between ARID1A and clinicopathological variables. We then carried out the prognostic analysis of ARID1A in TCGA subtypes. Finally, we screened patients by random sampling and propensity score matching method and performed multiplex immunofluorescence to explore the effects of ARID1A on CD4, CD8, and PD‐L1 expression in TCGA subtypes.We collected 1248 postoperative patients with gastric adenocarcinoma, constructed tissue microarrays, performed immunohistochemistry for Seven variables independently associated with ARID1A were screened out: mismatch repair proteins, PD‐L1, T stage, differentiation status, p53, E‐cadherin, and EBER. The independent prognostic variables in the genomically stable (GS) subtype were N stage, M stage, T stage, chemotherapy, size, and ARID1A. PD‐L1 expression was higher in the ARID1A negative group than in the ARID1A positive group in all TCGA subgroups. CD4 showed higher expression in the ARID1A negative group in most subtypes, while CD8 did not show the difference in most subtypes. When ARID1A was negative, PD‐L1 expression was positively correlated with CD4/CD8 expression; while when ARID1A was positive, this correlation disappeared.The negative expression of ARID1A occurred more frequently in the Epstein–Barr virus and microsatellite instability subtypes and was an independent adverse prognostic factor in the GS subtype. In the TCGA subtypes, ARID1A negative expression caused increased CD4 and PD‐L1 expression, whereas CD8 expression appeared independent of ARID1A. The expression of CD4/CD8 induced by ARID1A negativity was accompanied by an increase in PD‐L1 expression. Herein, we explored the prognostic and immune infiltration significance of ARID1A negative expression in TCGA subtypes in 1248 patients with gastric adenocarcinoma.22.1We collected 1347 patients with gastric adenocarcinoma who received initial surgical treatment at Weihai Municipal Hospital between January 2014 and December 2020. Subsequently, we excluded patients who received antitumor therapy before surgery . The tissue microarrays (TMA) were constructed as follows. First, two pathologists carefully reviewed the hematoxylin and eosin (H&E)‐stained slides of the screened patients and marked representative tumor areas. Second, we took out one tissue core (2 mm in diameter) from each corresponding formalin‐fixed and paraffin‐embedded (FFPE) donor block using a manual tissue sampling gun  and transferred the donor tissue cores to the recipient paraffin block . Finally, we fused the donor cores and the recipient block into TMAs by heating slowly to 65°C. After excluding the spots that did not contain tumor tissue and those that detached from the TMA slides, 1248 patients were included in the study.We obtained the following clinical pathological information by reviewing the electronic medical record (EMR) systems and pathological records: age, sex, tumor site, tumor size, differentiation status, WHO histological classification, Lauren classification, vascular invasion (VI), perineural invasion (PNI), invasion depth, lymph node metastasis, and distant organ metastasis. We restaged the enrolled patients according to the latest AJCC TNM staging system . Overall survival (OS) times was defined as the time from the date of diagnosis to the date of death or last follow‐up, which were obtained by EMR system or telephone follow‐up.This study was approved by the Ethics Review Board of Weihai Municipal Hospital (permission code: 2021053), which considered that the written informed consent from patients was unnecessary due to this study was retrospective and was conducted anonymously. The design and workflow of this study are shown in Figure 2.2We obtained sections of 2 μm thickness from TMA blocks and then implemented IHC staining for ARID1A, E‐cadherin, p53, PD‐L1, Ki‐67, HER‐2, MSH2, MSH6, MLH1, and PSM2 using an automated immunostaining machine  following the manufacturer's procedure. The primary antibodies' information were as follows, ARID1A , E‐cadherin , MSH2 , MSH6 , MLH1 , PMS2 , p53 , PD‐L1 , Ki‐67 , and Her‐2 . If the immunohistochemical staining of Her‐2 is 2+, further fluorescence in situ hybridization (FISH) is required. We detected EBV infection by the EBER‐ISH method using the EBER assay kits .2.3ARID1A was scored according to staining intensity and staining proportion of tumor cells, respectively. Staining intensity was scored as 0 (absence of any nuclear staining), 1 (faint nuclear staining), and 2 (intense nuclear staining); Staining proportion was scored as 0 (0%), 1 (1%–10%), and 2 (11%–100%). The above two scores were multiplied to obtain a final score, ranging from 0 to 4; 0 or 1 was defined as ARID1A negative, and ≥2 was defined as ARID1A positive.2.4We performed an analogous TCGA molecular classification according to the staining results of EBER, MMR, and E‐cadherin. First, we selected EBER‐positive cases as EBV subtype; Next, we classified the dMMR cases into MSI subtype based on the MMR result; Then, we classified cases with E‐cadherin negative expression as GS subtype; Finally, the remaining cases were classified as CIN subtype.2.5We randomly screened half of the ARID1A negative patients, followed by screening ARID1A positive patients using the propensity score matching (PSM) method in a 1:1 ratio. We then marked interest regions in tumor center (TC) and invasive margin (IM) on representative H&E‐stained slides. Finally, we constructed TMAs using the same method described above for subsequent mIF analysis.The mIF for CD4, CD8, PD‐L1, CK‐pan, and DAPI was performed at Tissuegnostics Asia Pacific Limited  using a five‐color kit from Tissuegnostics  following the manufacturer's protocol. Immunofluorescence images were acquired using the Tissue FAXS Cytometry platform of Tissuegnostics and were then high‐throughput quantitatively analyzed with Strata Quest software (version 7.0.1). The brief protocol was as follows: First, the fluorescence was detected by fluorescence microscope at different wavelengths in five channels respectively, dark blue for DAPI (TG470SN), skyblue for CK pan (TG430N), green for CD4 (TG520N), yellow for CD8 (TG570N), and red for PD‐L1 (TG650N). Next, the DAPI fluorescence staining channel was used for nucleus identification, and the parameters were adjusted by the “forward‐reverse tracking tools” according to the results of quantitative analysis to obtain the cell number in sight (n/sight). Then, the image segmentation of the nucleus and cytoplasm was performed, and optimal thresholds for the positivity of each marker were determined based on fluorescence signal intensity. Finally, each marker's positive cells (n/sight) were calculated according to the fluorescence signal around the nucleus.2.6p < 0.05 were then screened out for the subsequent multivariate logistic regression analysis (screening variables with the stepwise method) and least absolute shrinkage and selection operator (LASSO) regression analysis, respectively. Then, a random forest model was used to sort the variables filtered out by logistic regression and LASSO according to the importance, measured by increase in node purity (IncNodePurity), with higher values indicating more significant importance.p < 0.05 were then screened out for the subsequent multivariate Cox regression analysis and LASSO regression analysis, respectively. Then, a random survival forest model was used to sort the variables according to the variable importance (VIPM), with higher values indicating stronger predictive ability.t‐test  or Wilcoxon rank‐sum test was used to perform statistical analysis for continuous data in two groups. Spearman's rank test was used to analyze the correlation between immune markers . Two‐sided tests were adopted, and the differences were considered statistically significant when p < 0.05.All statistical analyses and statistical graphics were performed with R software (version 4.1.2). The chi‐square test was used to analyze the correlation between categorical variables, including the correlation between ARID1A and clinicopathological features, as well as the correlation between ARID1A and TCGA molecular classification. Variables with The R packages used in this study were listed as follows: ggplot2 (3.3.6), rcompanion (2.4.18), epiDisplay (3.5.0.1), MASS (7.3–54), forestmodel (0.6.2), glmnet (4.1–3), randomForest (4.7–1.1), survival (3.2–13), survminer (0.4.9), randomForestSRC (3.1.1), pheatmap (1.0.12), and MatchIt (4.3.1).33.1A total of 1248 patients were enrolled in this study with a median age of 64 years (range: 28.0–88.0), nearly 3/4  were male, and most of the lesions were located in the antrum . The distribution of AJCC TNM stage I, II, III, and IV in the cohort was 310 (24.84%), 299 (23.96%), 576 (46.15%), and 63 (5.05%), respectively. The main IHC findings were as follows: ARID1A negative in 275 patients (22.04%), EBER positive in 71 patients (5.69%), dMMR in 184 patients (14.74%), E‐cadherin absent in 418 patients (33.49%), Her‐2 positive in 79 patients (6.33%), and PD‐L1 positive in 87 patients (6.97%). The detailed clinicopathological information is shown in the “Total” column of Table The distribution of EBV, MSI, GS, and CIN subtypes in the entire cohort was 71 (5.69%), 181 (14.5%), 331 (26.52%), and 665 (53.29%), respectively Figure . Among t3.2p < 0.05 as follows: Size, T stage, M stage, TNM stage, differentiation status, Lauren classification, VI, Her‐2, PD‐L1, EBER, MMR, p53, and E‐cadherin  and a low expression category (blue predominant); this means the ARID1A negative group had higher expression of CD4, CD8, and PD‐L1 than ARID1A positive group Figure . The barWe analyzed the correlation between PD‐L1 expression in IHC and immune infiltration. The results showed that the PD‐L1 expression in IHC was consistent with that in mIF Figure . When AR4ARID1A mutation has been observed in various tumors, particularly gynecological tumors, such as 57% of ovarian clear cell carcinomasARID1A has also been identified as a novel driver geneARID1A gene.ARID1A gene contains many microsatellites.ARID1A gene exhibited more frequent short tandem repeats (STR) mutations, inferring MMR deficiency as the underlying cause of ARID1A mutations.ARID1A mutations preceded MSI.ARID1A was a causative gene for MSI rather than the target gene. A study confirmed that loss of ARID1A in EBV‐positive gastric cancer was partly regulated by EBV‐encoded miRNAsTP53, and simultaneous mutations in ARID1A and TP53 were rarely observed in the same tumor.In this study, we found that the proportion of ARID1A negative expression varied significantly among different TCGA molecular subtypes, with higher ARID1A negative proportion in the MSI subtype and EBV subtype, illustrating the close correlation of ARID1A with MSI and EBV, which is consistent with previous TCGA findings.Loss of ARID1A resulted in higher T stage, worse differentiation, and E‐cadherin deficiency in this study, which are well‐known factors contributing to poor prognosis in gastric cancer. Interestingly, loss of ARID1A is closely associated with MSI and EBV, which can lead to immune infiltration, activate the immune system to kill tumor cells, and appear to be a favorable prognostic factor in gastric cancer. Although some studies of gastric cancer suggested that alterations in ARID1A caused better survival,Some studies on gastric adenocarcinoma found SWI/SNF complex abnormalities to be an independent prognostic factor for OS in the GS subtype.+ TILs was confounded by MMR status.+ and CD8+ T cells in TCGA subtypes, implicating ARID1A involved in the immune infiltration through different mechanisms. Whether or to what extent the effects of ARID1A on TILs and PD‐L1 expression are influenced by confounding factors such as EBV and MMR is unclear, and more in‐depth investigations are still needed.It has been well documented that the loss of ARID1A caused an increase in tumor‐infiltrating lymphocytes (TILs) and increased expression of PD‐L1.+ T cells, leading to immune resistance and promoting tumor progression.+ T cells, suggesting that adaptive immune resistance is active and can be counteracted by inhibiting PD‐1/PD‐L1. Further hierarchical analysis found that PD‐L1 was highly expressed in gastric carcinomas with high CD8+ T infiltration only when ARID1A was negative. We speculated that the loss of ARID1A increased PD‐L1 expression by activating Akt signaling,PD‐L1 expressed on tumor cells can inhibit the function of CD8ARID1A mutation,Numerous studies of targeted therapies in a synthetic lethality manner have been conducted based on The limitations of this study are as follows. First, the TMA is an economical and highly efficient method for protein expression analysis, especially in large‐scale cohorts. However, there is a discrepancy between the results of protein expression obtained from TMA and the real results due to the heterogeneity of gastric cancer. Second, we used IHC and EBER‐ISH for TCGA classification instead of high‐throughput molecular techniques. However, protein expression may be insufficient to represent the complex molecular changes. Finally, we screened out a subset of patients for mIF using random sampling and PSM instead of the entire cohort, which may lead to biased statistical results.5+ T cells infiltration caused by ARID1A negativity was accompanied by the increase of PD‐L1 expression, which might induce adaptive immune resistance that can be resisted by inhibiting PD‐1/PD‐L1.In summary, we detected the IHC expression of ARID1A in gastric adenocarcinoma and explored the impact of ARID1A negative expression on prognosis and immune infiltration in combination with TCGA molecular classification. Negative expression of ARID1A occurred more frequently in the EBV and MSI subtypes and was an independent adverse prognostic factor in the GS subtype. The ARID1A negative group in the GS subtype may represent a particular population with the worst prognosis. The high CD8 expression caused by ARID1A negative was mainly due to MSI and EBV subtypes, whereas the high expression of PD‐L1 and CD4 resulted more from ARID1A itself. The increase of CD8Zhenkun Zhang: Conceptualization (supporting); data curation ; formal analysis ; investigation ; methodology ; validation ; writing – original draft (lead). Qiujing Li: Conceptualization (supporting); resources ; software ; validation . Shanshan Sun: Data curation ; formal analysis ; investigation ; software . Jing Ye: Conceptualization (supporting); resources ; validation ; writing – review and editing (supporting). Zhe Li: Data curation ; investigation ; writing – original draft (supporting). Zhengguo Cui: Methodology ; validation ; writing – review and editing . Qian Liu: Resources ; software . Yujie Zhang: Resources ; software . Sili Xiong: Investigation ; software . Shukun Zhang: Conceptualization (lead); data curation ; formal analysis ; methodology ; project administration ; supervision ; validation ; writing – review and editing .None.The authors declare no conflicts of interest.This study was approved by the Ethics Review Board of the Weihai Municipal Hospital (permission code: 2021053). The Ethics Review Board considered that informed consent for this study was not required due to its retrospective design.Table S1.Click here for additional data file.Table S2.Click here for additional data file.Table S3.Click here for additional data file.Table S4.Click here for additional data file.Table S5.Click here for additional data file.Table S6.Click here for additional data file.Table S7.Click here for additional data file.Table S8.Click here for additional data file.Figure S1.Click here for additional data file.


Lymphovascular invasion (LVI) is a crucial predictor of lymph node metastasis (LNM). However, few studies have investigated the LVI positivity rate and its clinical significance in pT1b esophageal squamous cell carcinoma (ESCC) using immunohistochemistry and elastin staining.We collected data from158 patients with pT1b ESCC who had undergone radical esophagectomy. All paraffin blocks of invasive carcinoma from each patient were subjected to HE staining, elastin staining + CK (AE1/AE3) immunohistochemistry (E&IHC), and CD31/D2-40 + CK (AE1/AE3) double immunohistochemistry (D-IHC). The LVI was classified into types, i.e., vascular invasion (VI) and lymphatic vessel invasion (LI), and its location, quantity, and clinical significance were explored.P < 0.001, respectively). CD31-VI and E-VI were independent adverse prognostic factors for recurrence-free survival (RFS), and they were significantly associated with poor distant metastasis-free survival and overall survival in pT1b ESCC. Intratumoral LVI was also crucial in pT1b ESCC, and L2 (the count of D2-40-LI was 5 or more) was the strongest predictor for LNM and RFS in pT1b ESCC.The positivity rates of VI by E&IHC (E-VI), VI by CD31D-IHC (CD31-VI), and LI by D2-40 D-IHC (D2-40-LI) were significantly higher than those obtained by HE staining (E&IHC and D-IHC can dramatically improve the detection rate of LVI in pT1b ESCC, and the classification and grading of LVI can help to improve the prediction of LNM and prognosis.The online version contains supplementary material available at 10.1186/s12885-023-10858-7. Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant tumors worldwide, particularly in East Asia. Superficial ESCC (SESCC) is defined as a tumor restricted to the mucosa or submucosa, irrespective of regional lymph node metastasis (LNM), and can be classified as either pT1a or pT1b. The extent of tumor invasion in pT1a is the lamina propria or muscularis mucosa, while tumor invasion in pT1b is restricted to the submucosa –4. The iLymphovascular invasion (LVI) is deemed an essential step in tumor metastasis and a significant predictor of metastasis. Many studies have shown that LVI is important for LNM in SESCC –13. Howe. demonstrated that intratumoral LI was not related to LNM. Only LI of the intramucosal and submucosal peritumoral areas was significantly associated with LNM in ESCC patients [On the other hand, most studies on LVI in ESCC have not attempted to differentiate between vascular invasion (VI) and lymphatic vessel invasion (LI) –16. Howepatients . In addipatients , and theTherefore, in the present investigation, all paraffin blocks containing infiltrating carcinoma from each patient were subjected to HE staining, E&IHC, and D-IHC to explore the actual LVI positivity rate in patients with pT1b ESCC. Additionally, we refined studies on LVI by focusing on its location and quantity and explored its clinical significance.One hundred fifty-eight patients with pT1b ESCC who underwent radical surgical resection using thoraco-abdominal lymphadenectomy at the National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College between February 1990 and January 2004 and had complete pathological and clinical data were evaluated. None of the patients received radiotherapy, chemotherapy, or biological therapy before surgery. The study protocol was approved by the Institutional Review Board of the National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College . All procedures were conducted in accordance with the ethical standards of the Declaration of Helsinki.The tumor lesions in each case were fully sampled for microscopic examination. We selected all paraffin blocks containing infiltrating carcinoma from each patient and then made serial 4 μm slices for HE staining, E&IHC and D-IHC.E&IHC was performed as follows. The slices were dewaxed, antigen retrieved, and subjected to immunohistochemistry with CK (AE1/AE3) . Next, improved aldehyde complex red staining was performed to detect venous elastic fiber . The elaFor D-IHC, sequential double immunohistochemical staining was performed using a BOND III Immunostainer (Leica Microsystems) and a Bond Polymer Refine Detection Kit (DS9800) as the first stain and a Bond Polymer Refine Red Detection Kit (DS9390) as the second stain. D2-40 and CD31  were visualized with DAB chromogen, and CK (AE1/AE3)  was visualized with Fast Red chromogen.Normal esophageal tissue was used for each batch of immunohistochemical staining to establish one external positive control and one negative control. In the negative control, buffers were used instead of primary antibodies. In addition, negative and positive internal controls are also present in each section.The results were independently interpreted by two pathologists (LL and BW). A multi-head microscope was used to reach a consensus in the case of inconsistent results. The presence of tumor cells in the lumen with endothelial cells with a gap between the tumor cells and vessel wall was judged as LVI. The presence of red blood cells in the lumen or smooth muscle around the vessel by HE staining was judged as VI and recorded as HE-VI Fig. A. The prWe recorded the tumor location , macroscopic type  , depth oP < 0.05 was regarded as statistically significant.The data were statistically processed using IBM SPSS Statistics for Windows, version 25.0 . The relationship between the clinicopathological variables and LNM was analyzed by logistic regression analysis. Receiver operating characteristic (ROC) curves were used to determine the cutoff points of counting variables, and the Kaplan–Meier method and log-rank test were used to analyze survival. The significance of clinicopathological markers relative to RFS, DMFS, and OS were analyzed using a Cox proportional hazards regression model . P < 0.001, respectively). Compared with HE staining, E&IHC and CD31 D-IHC increased the positivity rate of VI by 64.6% and 20.9%, respectively, and D2-40 D-IHC increased the detection rate of LI by 27%.Three hundred sixty-two paraffin blocks containing invasive carcinoma (1–6 paraffin blocks and an average of 2.3 blocks per patient) were selected. The positivity rates of HE-VI and HE–LI were 4.4% (7/158) and 22.2% (35/158), respectively. The positivity rates of E-VI, CD31-VI, and D2-40-LI were 69% (109/158), 25.3% (40/158), and 49.4% (78/158), respectively, which were higher than those of HE-VI and HE–LI (P = 0.001). D2-40-LI showed no significant correlation with LNM (P = 0.094). However, peritumoral D2-40-LI was significantly correlated with LNM (P = 0.015). The count of D2-40-LI of all paraffin blocks was 1 to 73 in the positive case of LI. A ROC curve was drawn to obtain the optimal cutoff point of the D2-40-LI count for predicting LNM, which was 4.5 . According to the D2-40-LI count of all paraffin blocks, D2-40-LI was subdivided into three groups, L0 (0), L1 (1–4), and L2 (≥ 5). There was a statistically significant difference between L2 and L0 for LNM but not between L1 and L0  experienced recurrence between 1 and 178 months, with a median recurrence time of 29.5 months. Nineteen patients (12.3%) developed distant metastasis between 1 and 192 months, with a median time of 31 months. The lung was the most common site of distant metastasis, accounting for 36.8% of cases.P < 0.05; Table P < 0.05; Table P < 0.001).HE–LI, E-VI, CD31-VI, D2-40-LI, L2, tumor location (upper thoracic), and LNM were significantly associated with RFS and OS  was slightly lower than that of all paraffin blocks containing infiltrating carcinoma (OR: 6.889) Table S.VI and LI were classified as intratumoral or peritumoral based on their location. As shown in Table Pathologists generally use separate immunohistochemical analyses of vasculature markers in daily work to identify LVI. However, it is difficult to determine whether the vessels contain tumor cells in some cases. In our study, adding epithelial markers made our study results more accurate. Using E&IHC and D-IHC for all blocks containing invasive carcinoma from each patient to evaluate LVI could better represent the true incidence of LVI. E&IHC and D-IHC significantly improved the detection rate of LVI and distinguished the type more accurately.For VI detection, the sensitivity of HE staining was low (just 4.4%), and it is necessary to carry out E&IHC and D-IHC simultaneously. VI shown by D-IHC and E&IHC revealed different blood vessels. E-VI is characterized by thick-walled venous vessels with elastic fiber. Because of tumor invasion, no apparent lumen structure is observed, and vascular endothelial cells are destroyed; therefore, detection by HE staining or IHC is challenging. Additionally, VI sometimes resembles the spaces formed by cancer nest contraction with HE staining Fig.  C. Howev. mainly analyzed in detail the importance of peritumoral LVI [In breast cancer, because few examples of intratumoral LVI without concomitant peritumoral emboli exist, Lee A K et aloral LVI . The litoral LVI . Thus, Loral LVI . However. divided LI into four groups according to the number of LI  in the IHC analyses [In the present study, both HE–LI and peritumoral D2-40-LI were significantly correlated with LNM. However, there was no significant correlation between D2-40-LI and LNM. We believe that the reason for the difference is due to the sensitivity of D-IHC to LI, which is significantly higher than that of HE staining, thereby greatly increasing the D2-40-LI positivity rate. Therefore, D2-40-LI was further grouped according to its count. L2 (the count of D2-40-LI was 5 or more) was significantly associated with LNM, RFS, DMFS, and OS, which had a larger Odds ratio and HR than HE–LI Tables , and 3. analyses . They sh. suggested that LVI was the only independent factor of disease-free survival in patients with sm2 and sm3 lymph node-negative SESCC, but LVI was not found to have a stronger prognostic impact than LNM [The literature suggests that ESCC patients with LVI have a poor prognosis. However, insufficient studies exist on the relationship between LVI and the prognosis of SESCC patients , 35. Oguthan LNM . In our The limitation of the study was that it was retrospective, and all patients had undergone thoraco-abdominal lymphadenectomy. Patients with sm3 accounted for a large proportion (107/158), and a certain degree of bias existed in the correlation analysis of the depth of invasion.In conclusion, E-VI and CD31-VI were associated with DM and a poor prognosis. The D2-40-LI count is more important than the location, and high-frequency D2-40-LI has a better predictive value for LNM and RFS. In terms of the count of D2-40-LI, it is not necessary to analyze all paraffin blocks of invasive carcinoma if the pathologist can determine that the case belongs to the high-grade group (the count of D2-40-LI was 5 or more). We recommend using elastin staining and CD31/D2-40 IHC to identify LVI in all patients with pT1b ESCC based on the unoptimistic LNM rates and recurrence rates. Although E&IHC and D-IHC are more expensive and complicated, they are recommended if feasible due to their more clarity.Additional file 1:Table S1. Relationshipbetween the D2-40-LI of one representative paraffin block (RD2-40-LI) and lymphnode metastasis (LNM), recurrence-free survival (RFS), overall survival (OS)and distant metastasis-free survival (DMFS) of pT1b ESCC patients.Additional file 2:Figure S1. The survival curves of 154 patients with pT1b esophageal squamous cellcarcinoma stratified by the count of D2-40-LI of one representative paraffinblock (RD2-40-LI).


Both carvedilol and flecainide effectively suppressed OT PVCs in patients without structural heart disease, with flecainide showing a superior efficacy compared to carvedilol.The mechanism of premature ventricular complexes (PVC) occurring in the ventricular outflow tract (OT) is related to an intracellular calcium overload and delayed afterdepolarizations that lead to triggered activity. The guidelines recommend using beta-blockers and flecainide for idiopathic PVCs, but they also acknowledge the limited evidence supporting this recommendation. We conducted a multicenter, randomized, open-label pilot study comparing the effect of carvedilol and flecainide on OT PVC, which are widely used to treat this arrhythmia. Patients with a 24 h Holter recording a PVC burden ≥ 5%, which showed positive R waves in leads II, III, and aVF, and without structural heart disease were enrolled. They were randomly assigned to the carvedilol or flecainide group, and the maximum tolerated dose was administered for 12 weeks. A total of 103 participants completed the protocol: 51 with carvedilol and 52 with flecainide. After 12 weeks of treatment, the mean PVC burden significantly decreased in both groups: 20.3 ± 11.5 to 14.6 ± 10.8% with carvedilol ( Premature ventricular complexes (PVC) occurring in a structurally normal heart are called idiopathic PVCs, and they commonly originate from the ventricular outflow tract (OT) myocardium ,2. OT PV+/Ca2+ exchangers and finally resulting in delayed afterdepolarizations (DADs) that lead to triggered activity. Among the various beta-blockers, only carvedilol is known to have the potential to directly inhibit the release of SOICR in vitro [Carvedilol is one of the most effective beta-blockers for reducing ventricular arrhythmias and mortality in patients with heart failure ,6. In adin vitro ,8. The min vitro , and triWe hypothesized that carvedilol would be more effective for OT PVCs than other beta-blockers due to its direct SOICR inhibition effect. Therefore, we aimed to demonstrate its effect through a multicenter, randomized, open-label pilot study. In addition, a randomized, direct comparison with flecainide was planned. Although flecainide is a well-proven class Ic antiarrhythmic agent and is already widely used for treating OT PVCs, there has been no structured, randomized trial to demonstrate its efficacy for OT PVCs.This was a multicenter, randomized, open-label pilot study comparing the effect of carvedilol with flecainide on OT PVCs conducted in 11 tertiary hospitals in South Korea. Data collection adhered to the principles outlined in the Declaration of Helsinki (2013) and Good Clinical Practices. The ethics committees and corresponding health authorities approved the protocol for all sites. All the patients provided written informed consent before enrollment. This trial was registered with ClinicalTrials.gov, number NCT03587558.(1)Pregnant subjects, subjects planning for pregnancy, or breastfeeding women (nursing mothers);(2)History of coronary artery disease;(3)History of hypersensitivity to carvedilol;(4)Subjects with cardiogenic shock;(5)Subjects with a significant atrioventricular block (AV block) (2nd or 3rd degree) or sinoatrial node dysfunction;(6)Subjects with cardiomyopathy with or without reduced LV EF;(7)Presence or history of respiratory diseases with bronchospasms, such as asthma and chronic obstructive pulmonary disease;(8)Receiving any medications known as monoamine oxidase inhibitors;(9)Subjects with severe hypotension displaying systolic blood pressure ≤ 90 mmHg;(10)Subjects with severe hepatic impairment;(11)Asymptomatic PVC or non-sustained ventricular tachycardia (NSVT) following myocardial infarction;(12)Subjects with any atrial fibrillation.Individuals who met the following criteria were included in the study: (1) between the ages of 19 and 80 years with no previous history of treatment for PVCs; (2) had symptomatic OT PVC, which was confirmed by positive R waves in leads II, III, and aVF of electrocardiography (ECG); (3) over 5% of PVC burden  ; (4) norRandomization was performed by an independent statistician at a ratio of 1:1 based on the sequence and was stratified according to the trial site using the SAS system randomization program .Eligible patients were randomized into either the carvedilol group or the flecainide group. Participants assigned to the carvedilol group received a once-daily dose of a sustained-release formulation of carvedilol. In South Korea, an 8/16/32/64 mg sustained-release formulation, once-daily dose of carvedilol is available and equivalent to 3.125/6.25/12.5/25 mg twice daily. Participants assigned to the flecainide group received a twice-daily dose of flecainide as the conventional method. The dosages of the medications were titrated up to the maximum based on the patient’s tolerance and the occurrence of side effects, which were evaluated every two weeks. The assessment of whether the dosage of the drug has reached the maximum was left to each physician’s discretion.(1)Visit 1 (Baseline): Informed consent was achieved, and confirmation of inclusion/exclusion criteria with randomization was performed. Information including body weight, gender, age, name , and concomitant diseases was collected from subjects. Physical examination and heart rate/blood pressure measurement while sitting were carried out. The results of routine 12-lead ECG, echocardiography, treadmill test, and PVC burden on 24 h Holter monitoring were investigated. Patient-reported symptoms, assessed on a visual analog scale , were also recorded.(2)Visit 2 (2 weeks ± 7 days): The following data were collected: heart rate and blood pressure while sitting, routine 12-lead ECG, VAS score, and medication compliance. Patients were asked whether any adverse events had occurred and to grade the event, if any.(3)Visit 3 (4 weeks ± 7 days) and Visit 4 (6 weeks ± 7 days): These visits were optional. However, according to the attending doctor’s discretion, more frequent visits could be made for drug dosage optimization.(4)Visit 5 (3 months ± 7 days from when a subject reaches their maximum tolerable dose): The measurement of the heart rate/blood pressure while sitting was carried out. In addition, the results of routine 12-lead ECG and PVC burden on 24 h Holter monitoring were collected. Patient-reported symptoms, assessed on a VAS, were also recorded.The patient’s tolerance and occurrence of hypotension and bradycardia were mainly assessed in the carvedilol group. In the flecainide group, QRS widening was intensively monitored. After reaching the maximum tolerated dose, the drug was maintained for three months. Baseline clinical and demographic data were collected along with the patients’ 24 h Holter monitoring data. Interim outpatient clinic visits were also left to the physician’s discretion, but the following schedule was recommended:(1)①Beta-blockers;②Non-dihydropyridine calcium channel blockers: diltiazem, verapamil;Antihypertensives other than carvedilol, especially the following drugs:(2)Monoamine oxidase inhibitors;(3)Antiarrhythmic drugs, including digitalis;(4)Other drugs that might affect patient safety or analysis of the study’s outcome in the opinion of the investigator.Any of the following medications were not taken during the study period:The primary outcome was an absolute reduction in the PVC burden after three months of treatment. The secondary outcome was patient-reported symptoms assessed on a VAS. The primary and secondary outcomes were compared between the carvedilol and flecainide groups. The safety outcomes were assessed at each visit and included participant-reported adverse events. A serious adverse event was defined as any medical event that resulted in death, was life-threatening, required hospitalization, or caused substantial disability or incapacity. A severe adverse event was any event that interfered with the patient’s usual function, as deemed by the investigator on the case report form.Until now, no randomized study has evaluated the efficacy of carvedilol and flecainide on OT PVCs. Therefore, we conducted a pilot study, and sample size calculation was not performed. The researchers aimed for 100 participants (about 50 patients for each group) because it was felt that this would be a large enough sample for the pilot trial.t-test and chi-square test were used for continuous and categorical variables if normality was accepted. If the sample did not meet the normality assumption, the following method was used: the Mann–Whitney test was used to compare within-group continuous variables before and after the intervention, and the Wilcoxon signed-rank test was used to analyze the differences and changes in values between the two groups. Statistical analyses were performed using the MedCalc Statistical Software  or SAS . A p-value < 0.05 was considered statistically significant.Continuous variables are expressed as the mean value ± standard deviation or interquartile range when the values do not follow a normal distribution. Categorical variables are expressed as numbers and percentages. The paired/independent sample The corresponding author designed the trial. The sponsor, Chong Kun Dang Pharmaceutical Corporation, did not contribute to the trial design, conduct, oversight, data analysis, or manuscript writing. The first two authors and the corresponding author mainly wrote the manuscript. All authors reviewed and approved the final draft. The sponsor covered all costs associated with the trial, including the cost of the carvedilol and all tests that were not otherwise clinically indicated for the trial’s purposes. Most data analyses were performed by a clinical research organization (Seoul CRO). All events were documented from sources, including, but not limited to, the paper and electronic charts, the laboratory, and imaging test reports, and were adjudicated by an independent clinical events committee, whose members were unaware of the trial-group assignments. Serious adverse events were reviewed by an independent data and safety monitoring board according to a predefined schedule.n = 51) or the flecainide (n = 52) groups. The participants’ baseline demographic and clinical characteristics are shown in From 20 June 2018 through 9 June 2020, 103 patients were recruited and randomly assigned to the carvedilol (Most patients in the carvedilol group received an 8 mg (41.2%) or 16 mg (52.9%) dose, and in the flecainide group, 69.2% received 100 mg and the rest received 150 mg or 200 mg (28.8%). Blood pressure, heart rate, PR interval, and QRS duration changes after treatment are shown in p < 0.0001) and 17.1 ± 9.9 to 6.6 ± 9.9% in the flecainide group (p < 0.0001) (p = 0.023)  . The mea= 0.023) .The used dosages and the efficacy on PVC reduction according to each dosage of the drugs are shown in p < 0.0001) and 4.1 ± 2.2 to 2.0 ± 2.0% in the flecainide group (p < 0.0001) (p = 0.685). The major results of the study are summarized in The secondary outcome, symptom improvement, was assessed by a 10-item VAS scale, and it also decreased significantly in both groups after the treatment: 4.2 ± 2.1 to 2.1 ± 2.2% in the carvedilol group ( 0.0001) . The degn = 51): two involving dizziness, two involving dyspnea on exertion, one involving a headache, one involving fatigue, one involving palpitation, and one involving sexual dysfunction. In the flecainide group, minor adverse events were as follows (n = 52): one involving dizziness, two involving headaches, one involving a burning sensation, one involving somnolence, one involving fatigue, and one involving blurred vision.No serious adverse events were observed in either treatment group during the study period. Minor adverse events in the carvedilol group were as follows , and phospholamban . These rHowever, in case of excess intracellular calcium concentration, there is increased activity of the SR calcium pumps (SERCA). The SERCA increases its activity to remove calcium from the cardiac cellular myoplasm into the SR. When the SR reaches a critical threshold, it cannot accommodate such calcium overload and spontaneously releases some of the calcium back into the myoplasm through RyR2 after repolarization and during diastole; this process is called SOICR . The eleThese hypothesized mechanisms are supported by the fact that when using therapeutic agents that antagonize the mechanisms, OT PVCs are suppressed. Beta-blockers terminate tachycardia by antagonizing the arrhythmogenic effects of adrenergic stimulation. Calcium-channel blockers diminish the slow inward calcium current and thus terminate the arrhythmia. Valsalva maneuvers release acetylcholine, which inhibits the production of adenylyl cyclase and cAMP, thereby lowering the level of intracellular calcium. Adenosine acts in a similar manner to acetylcholine to reverse intracellular calcium overload . Given tPVCs are extremely common, and we frequently encounter patients with symptomatic OT PVCs in the clinic. As with previous guidelines, the latest guidelines also recommended the use of beta-blockers and flecainide for the treatment of idiopathic PVCs. However, guidelines also indicated that the evidence of this medical treatment is scarce . Most stRecently, Tang and colleagues published a paper regarding medical therapy for frequent idiopathic PVCs . This stOn the other hand, a retrospective study comparing RFA to medical therapy over a follow-up period of 6–12 months demonstrated reductions in PVC burden of 36% with beta-blockers and of 82% with antiarrhythmic drugs . This st2+ can activate the inward Na+-Ca2+ exchange current, causing a DAD [Carvedilol seems to have a more significant antiarrhythmic potential than the other beta-blockers ,6,17. Kang a DAD . Indeed,ng a DAD ,23. Amonng a DAD . With thCarvedilol is a non-selective beta-blocker, and its SOICR inhibitory effect has been experimentally proven. It has shown a superior antiarrhythmic effect compared to metoprolol in large-scale clinical studies of heart failure. Therefore, we assumed that carvedilol would show a distinguished efficacy on OT PVC inhibition compared to other beta blockers. However, the results of our study using carvedilol were not much different from the results of the observational study using bisoprolol  or the MIn our study, after three months of treatment, PVC burden was reduced by about 60% with flecainide and 25% with carvedilol. In particular, flecainide significantly suppresses OT PVCs from a dosage of 50 mg bid, and at higher dosages, it almost completely inhibits OT PVCs. Hence, flecainide showed superior antiarrhythmic effects, which was expected since it is a well-proven class Ic antiarrhythmic agent. However, one of our study goals was to verify the antiarrhythmic effect of carvedilol as described above, and contrary to our expectations, carvedilol showed only modest effects.Although carvedilol did not show the efficacy that we expected in treating OT PVCs, it is certain that it significantly reduced the burden and symptoms of OT PVCs. The clinical meaning of our study can be summarized as follows. Firstly, no studies have systematically evaluated the treatment of idiopathic OT PVCs using carvedilol and flecainide. Furthermore, no studies have compared these two drugs in a randomized fashion. Through our study, the effects of carvedilol and flecainide on idiopathic OT PVCs were quantitatively and qualitatively evaluated. Second, carvedilol has well-proven clinical benefits and safety in patients with heart failure, and class Ic antiarrhythmic medications are generally avoided due to concerns for proarrhythmic/negative inotropic effects in patients with heart failure. Therefore, we propose that carvedilol can be selected as the preferred medication for the treatment of OT PVCs in patients with heart failure. In addition, it can be used safely when the patient’s heart function is not evaluated.Meanwhile, a recent study suggested that flecainide also has an RyR2-inhibitory effect which can suppress SOICR . MoreoveIn an in vitro study, the concentrations of carvedilol required to suppress the SOICR (0.3–1 μM) were much higher than those needed for a beta-blockade (~1 nM) . Therefop = 0.790). In the flecainide group, a standard/high dose was used in 28% of the patients . The differences in drug dosages between the two drugs may have affected our experimental results. Of note, there was no difference in PVC burden reduction between the groups using 8 mg and 16 mg of carvedilol; the exact reason for this is unclear. Indeed, on the other hand, it is noteworthy that even at lower doses, carvedilol showed some antiarrhythmic effects. This suggests that lower doses of carvedilol may still be effective in treating OT PVCs and may have potential benefits in reducing the risk of adverse events associated with higher doses. Further studies are needed to determine the optimal dose of carvedilol for this indication.In our study, the carvedilol doses administered were as follows: 8 mg in 22 patients, 16 mg in 27, 32 mg in 2, and 64 mg in 1. Most patients used 8 mg or 16 mg, which are relatively low doses, and the degree of the reduction in the PVC burden did not significantly differ between these two doses: carvedilol 8 mg −6.1 ± 6.4% and carvedilol 16 mg −5.4 ± 11.5%, To date, many research results have been published on the mechanism of OT PVCs and pharmacological/non-pharmacological treatment methods. However, there are still unresolved questions: (1). What is the mechanism of OT PVCs that occurs independently of sympathetic activation, such as during rest or sleep? (2). Why does DAD occur only in the OT myocardium due to sympathetic activation? Moreover, is there a problem with the RyR2 in the OT myocardium, or can other receptors’ abnormalities also occur in the OT myocardium? (3). Does carvedilol only act on abnormal RyR2, or could it also act on normal RyR2? Interestingly, to further complicate the issue, as described in the study by Tang et al. , a 32.7%We used 24 h Holter monitoring to assess PVC frequency in our study, but recent evidence has demonstrated that substantial daily variation may occur . TherefoOne of the significant limitations of our study is that we did not strictly control drug dosages and left them to the physicians’ discretion. This could have led to variations in the efficacy of the drugs, and the interpretation of the results should take this into account. Indeed, the reasons for determining the maximally tolerated dose should have been investigated in detail. Notably, a relatively low dose of carvedilol was used compared to flecainide. One possible explanation could be that physicians may have chosen to use lower doses to avoid potential side effects such as hypotension or bradycardia, which can occur with higher doses of carvedilol. This may have affected the research results, but it should be noted that while lower doses may have been used in some patients for the reasons mentioned above, the optimal dose of carvedilol for the treatment of OT PVCs has not yet been established and may vary depending on individual patient characteristics. Therefore, further studies are needed to determine the most effective and safe dosage of carvedilol for this indication.Finally, although we performed randomization, this study was a pilot study with a small sample size, making it difficult to draw definitive conclusions.In our study, both carvedilol and flecainide effectively suppressed idiopathic OT PVCs, but there was a difference in the degree of PVC suppression between the two drugs. We hope that our study results can contribute to further research in this field and encourage more comprehensive studies with larger sample sizes and stricter control over drug dosages.


Ms5.0 earthquake and Menyuan Ms6.9 earthquake are the two Ms ≥ 5.0 earthquakes in recent years. The spatio-temporal evolution of Rn across the fault before the two Ms ≥ 5.0 earthquakes were explored by combining a solid seismogenic model and numerical simulation results in this study. The results demonstrates the spatial distribution of Rn concentration intensity varies over time, indicating the evolving characteristics of fracture zone activity. The time-series variation characteristics are closely related the Zhangye Ms5.0 earthquake and Menyuan Ms6.9 earthquake. Overall, in the seismic source area and surrounding medium area of Zhangye Ms5.0 earthquake, the soil gas Rn anomaly across faults characterized by a turning upward trend after continuous decline. The closer to the source area, the more obvious the upward trend. For Menyuan Ms6.9 earthquake, the survey line (HT1) located in the main fracture zone of the earthquake and the survey line  closer to the epicenter also showed a similar trend, while the other measurement lines in far-field exhibit declining trend before the Menyuan Ms6.9 earthquake. Therefore, the continuous decline trend of soil gas may be crucial information for medium-term earthquake preparation in the seismogenic zone, and the trend of turning upward after continuous decline is a significant signal of short-term seismogenic event in far-field. This research could improve the understanding of the anomalous features of soil gas precursors and tracking the active sections of the fault. According to the model, the earthquake area canseismic source area, the surrounding medium area be divided into three sections: the seismic source area, the surrounding medium area, and the fracture fragmentation area.The mid-eastern segment of the Qilianshan fault zone (QLF) on the northeastern margin of the Qinghai–Tibet Plateau is considered one of the key seismic hazard areas. The Zhangye  Soil gas geochemical properties of deep gases discharged in geologically active areas has been extensively employed for precursors of tectonic 15. The preparation and occurrence of earthquakes encompass a complex physical and chemical process, involving the energy transfer of deep materials, changes in medium conditions, and interactions between underground fluids, such as water and gas, and rocks under stress17. This process necessitates the migration, differentiation, and evolution of fluids18. Active fault zones serve as channels for underground fluid escape and are crucial locations for earthquake preparation and occurrence24. The gas geochemical properties of active fracture zones are often linked to the region's seismic activity27. In fact, these properties have been successfully utilized to elucidate key earthquake mechanisms along major active fault zones in Taiwan, Japan, and China33. Soil gases  in fracture zones frequently exhibit anomalies before and after earthquakes34, with the magnitude of these anomalies often fluctuating alongside seismic activity35. Research on the relationship between released CO2 fluxes and seismic activity in the L'Aquila region and the Apennines in Italy has demonstrated that CO2 gas released through rupture zones plays a crucial role in earthquake nucleation, occurrence, and aftershock activity36. Continuous monitoring of soil gas Rn concentrations on the Muzaffarabad fault in Pakistan abnormal Rn concentrations approximately 30 days prior to seven earthquakes, with magnitudes ranging from 0.8 to 4.9, impacting the rift37. Subsequent to the Wenchuan Ms8.0 earthquake, anomalies in soil gas He, CH4, Rn, H2, and Hg concentrations were found to be closely associated with surrounding aftershocks during drilling in the seismogenic rupture zone by scientific drilling holes No.1 (WFSD-1) and No. 2 (WFSD-2)39. Emission of CO2 was observed in association with earthquakes at the Lassen Peak volcano   and the Eger Rift (Czech Republic), indicating close connections between the Earth's surface and its crust by fluid transport. 575 earthquakes occurred at Cava dei Selci  from 2009 to 2021 showed that increase of CO2 flux seemed related to extensional deep earthquakes40. Additionally, the soil gas He and H2 anomalies in the Longmenshan rupture zone were observed to decrease as aftershock intensity diminished.Subsurface fluids constitute essential components of the Earth system, playing a critical role in Earth’s evolution as an effective medium for interconnecting the planet's various layersMs6.9 earthquake  occurred in Menyuan County, Haibei Prefecture, Qinghai Province, China, with a focal depth of approximately 10 km. Additionally, the Zhangye Ms5.0 earthquake took place in the middle western section of the Qilian Mountain seismic belt on September 16, 2019. This study aims to investigate the spatial and temporal evolution of cross-fault Rn concentrations prior to two Ms ≥ 5.0 earthquakes in order to explore effective information on earthquake precursors. The research could enhance our understanding of the anomalous features of soil gas precursors and facilitate tracking of active fault sections.It is evident that underground fluids have gained increasing prominence in earthquake prediction and related studies in recent years. However, due to difficulty in natural earthquake prediction, research on combining earthquake monitoring results with physical prediction mechanisms is still very scarce. Most observations on seismic geochemical effects lack physical mechanism support for identifying anomalies. This is the key to break through the extraction of fault Soil gas earthquake anomaly precursors. The mid-eastern region of the Qilian Mountains Fault (QLF) is considered a key hazard area in Mainland China. Since 2016, six periods of Rn concentration data have been gathered from nine measurement lines deployed across the Middle East section of the QLF. On January 8, 2022, an 42. This region is characterized by tectonic deformation and intersected by numerous active faults, rendering it one of the most seismically active and intense areas in mainland China, with substantial crustal movements46. The primary active fracture zones in this study area include the Sunan-Qilian fault, the Yumushan fault, the Minle-Damaying fault, the Huangcheng-Shuangta fault, and the Lenglongling fault, all of which are crucial components of the Qilian Mountains' northern margin  measurements were conducted at nine survey sites  situated on various NWW-oriented faults running parallel to each other and in oblique rows, covering the mid-eastern region of the Qilian fault Table .Table 1L61. No extreme climatic variations were observed during the monitoring period, and the meteorological conditions at each measuring point remained relatively stable.To minimize the impact of meteorology on soil gas concentrations, surveys were conducted under stable meteorological conditions in June of each year from 2016 to 2021. Rn concentrations, temperature, humidity, and atmospheric pressure were simultaneously measured using an AlphaGUARD detector . According to the detector's requirements, 15 to 20 values were measured during each assessment. All samples were collected using a stainless-steel probe inserted into the ground at a depth of 80–100 cm, depending on soil consistency and thickness, in order to minimize the effects of meteorological variablesIn this paper,the concentration strength of each survey site was calculated.Concentration intensity refers to the measurement of the change degree of the of fault gas concentration near the fault plane relative to the background concentration. It can intuitively reflect several characteristics of fault media such as porosity, fragmentation, and fracture development, as well as the mechanics and motion state of fault activity It is the main characteristic parameter for analyzing the spatial characteristics of fault gas.The calculation formula is as follows:Concentration strength (S) = Anomaly threshold/background value.S_max). This approach utilizes the maximum abnormal concentration values to calculate concentration intensity, better explaining the peak value of the concentration intensity for each profile. Additionally, this method minimizes differences in site-effect factors that influence gas release concentrations, such as soil and rock properties63. Details of the nine survey lines are listed in Table The background value is obtained by calculating the mean value of the remaining data after removing jump values, reflecting the normal accumulation level of specific elements or components in a given fault zone. The anomaly threshold can be expressed as the background value plus ‘n’ standard deviations n = 1, 2, 3,…); in this case, n = 2 was used. The purpose of this is to eliminate the measured value instability caused by environment. Calculations and evaluations led to the decision to use the maximum value method for determining the concentration strengthof soil gas Rn mm/a, and the left-sliding rate was (0.95 ± 0.11) mm/a25. The linear fitting results of the vertical sliding rate of the Minle-Damaying fault were (0.91 ± 0.09) mm/a41. The Huangcheng–Shuangta fault experienced significant activity in the Holocene, most notably the Gulang Ms8.0 earthquake in 192743. The Quaternary slip rate of the Lenglongling fault was determined to be (4.3 ± 0.7) mm/a by radiocarbon dating methods, while the slip rate since the late Holocene was (4.3 ± 0.36) mm/a49. The Huangcheng–Shuangta and Lenglongling fracture zones exhibit more activity than other fracture zones, indicating a relatively open fault. This corresponds well with the spatial distribution pattern of soil gas Rn in the QLF.The spatial distribution of seismic activity is illustrated in Fig. Ms6.9 earthquake on January 8, 2022, and the Zhangye Ms5.0 earthquake on September 16, 2019 have occurred. The seismogenic fault for the Menyuan Ms6.9 earthquake is the western segment of the Lenglongling fault, which ruptured to the Tolashan fault on the west side of its northwest end69, while the Zhangye Ms5.0 seismogenic fault is the northern margin fault of the Qilian Mountain (Sunan Qilian section).The Rn trend changes before and after 2019 have captured our particular interest, as they may be related to the Zhangye Ms5.0 earthquake, it has been observed that strong earthquakes typically do not occur in the high seismicity zones of fault systems, but rather in seismic gaps72. Subsequently, the concept of seismic gaps was introduced, and a solid seismogenic model was established to elucidate the conditions, processes, and associated precursors of strong earthquakes, as well as their spatial–temporal evolution74. This model is supported by a wealth of evidence from deep structures, mechanical analyses, rock fracture experiments, numerical simulation results, and observed facts75. According to the model, the earthquake area can be divided into three sections: the seismic source area, the surrounding medium area, and the fracture fragmentation area  and Dilatancy Hypothesis model (DD) have been proposed. However, these models mainly address the issue of epicenter precursors, and they cannot explain the complex spatiotemporal evolution process of precursorskes Ms ≥ .0, it hakes Ms ≥ .0, it haMs5.0 earthquake, the Rn concentration intensity of HT2 (27km away from the epicenter) decreased significantly from 2016 to 2017 and increased significantly from 2017 to 2019. In HT3 (2 km away from the epicenter), the Rn concentration intensity decreased from 2016 to 2018 and increased significantly from 2018 to 2019. Other survey lines also demonstrated some trend anomalies, but the short-term anomalies (turning upward trend after continuous decline) were not more pronounced than in HT2 and HT3. For the Menyuan Ms6.9 earthquake, HT1, HT4, and HT7 also displayed turning upward trends after continuous decline before the earthquake. HT1 is located on the the northern edge of the Qilian Mountains, the southern section of Sunan, 200 km from the epicenter. Despite this distance, it is situated along the main fault at the northern margin of the Qilian fault. The Lenglongling fault zone, where the Menyuan Ms6.9 earthquake occurred, is also an integral component of the northern Qilian fold belt in terms of geotectonics. Several aftershocks with a magnitude of Ms ≥ 3.0 were recorded in the region following the Menyuan Ms6.9 earthquake. Furthermore, except for three lines, all other measurement lines exhibited a continuing decline trend before the Menyuan Ms6.9 earthquake. The Menyuan Ms6.9 seismic activity and tectonic deformation directly expose the adjustment and transmission of tectonic deformation throughout the Qilian Mountain fault zone. As the survey lines did not directly cross the seismic fault of the Menyuan Ms6.9 earthquake, the abnormal features were not as pronounced as those observed in the Zhangye Ms5.0 earthquake.This model effectively explains the relationship between two Ms ≥ 5.0 earthquakes in the QLF. For the Zhangye The response of soil gas Rn to seismic events is not only related to the magnitude and distance from the epicenter but also controlled by the tectonic stress of deep major faults. Thus, the present observation results and the analysis of seismic cases support the body seismogenic model and numerical simulation computation results. This model elucidates the relationship between tectonic geochemical characteristics and rupture blockage, offering theoretical support for determining the time, space, and intensity of seismogenesis. The robust body seismogenic model can clearly explain the relationship between tectonic geochemical features and fracture locking, providing theoretical support for estimating the time, space, and intensity of earthquake nucleation.From a spatial distribution perspective, in 2016, the highest concentration intensity was observed in the middle (HT5) and west section (HT1). From 2017 to 2020, the spatial distribution characteristics of concentration intensity were stronger in the east than in the west.In 2021, the spatial distribution was less distinct, with values across the entire fault zone uniformly. The spatial distribution of Rn concentration intensity varies over time, indicating the evolving characteristics of fracture zone activity.Ms5.0 and Menyuan Ms6.9 earthquakes. For the Zhangye Ms5.0 earthquake, the Rn concentration intensity in major survey lines (except HT8 and HT9) showed a noticeable "V" temporal evolution trend, with an continue decline and then turn upward. For the Menyuan Ms6.9 earthquake, HT1 (located on the the northern edge of the Qilian Mountains) and HT4, and HT7 also displayed turning upward trends after continuous decline before the earthquake.While the other measurement lines exhibit a continued declining trend before the Menyuan Ms6.9 earthquake.From a temporal evolution trend before earthquake,the soil gas Rn concentration intensity across mid-eastern of the Qilian fault zone(QLF) exhibited a significant response to the Zhangye These observational facts were supported by the sturdy body seismogenic model and numerical simulation results. Consequently, we hypothesized that the continuous decline trend of fault gas may serve as a reliable indicator of the interlocked section of fault tectonic activity, and the trend of turning upward after continuous decline may represent a significant signal of a medium and short-term seismogenic event in the source area. Furthermore, the sturdy body seismogenic model could potentially provide theoretical support for determining the time, space, and intensity of seismogenesis based on the relationship between the tectonic geochemical characteristics and rupture locking.In this study, we explored the spatial–temporal evolution characteristics of soil gas Rn from 2016 to 2021 in the mid-eastern of the Qilian fault zone (QLF) before Zhangye Ms5.0 earthquake and Menyuan Ms6.9 earthquake by combining a solid seismogenic model and numerical simulation results. By analyzing the spatial–temporal evolution characteristics of soil gas Rn before two Ms ≥ 5.0 earthquakes in the Qilian fault zone, the following conclusions can be drawn:In summary, seismic prediction remains a difficult global scientific problem, and fluid geochemistry is one of the potential tools for earthquake prediction. The extraction of earthquake precursor information from soil gas must be based on a specific physical mechanism of seismogenesis. The results of long-term research have shown that, by studying the physical mechanisms of forecasting, the sensitivity and convenience of fault gas can be used to trace locked segments directly on dangerous faults for the identification and determination of precursor information, which can compensate for the limitations of springs and well-exposure locations used by subsurface fluid.


Dear Editors,FROM THE TEXT—“Unfortunately, despite the subsequent relevant events in Fukushima, federal funding in this area has declined significantly from its initial levels”RNCP REBUTTAL—It is not correct to say that federal funding in this area has declined significantly from initial levels. When the RNCP was established in 2004, funding via a congressional appropriation to the NIH Office of the Director was set at $45.93 million per year. As of the writing of this letter, the RNCP Fiscal Year 2023 budget is $52.64 million, all of which is earmarked for high dose radiation research.FROM THE TEXT—Acknowledging the loss of expertise within the field, several large projects funded by NASA have included aspects of training as a part of their research program, as did the National Institute of Allergy and Infectious Diseases/Biomedical Advanced Research and Development Authority, Centers for Medical Countermeasures against Radiation program in its initial 5 years of funding.RNCP REBUTTAL 1 – It is a mischaracterization to state that the Biomedical Advanced Research and Development Authority  is a part of the National Institute of Allergy and Infectious Diseases’ Centers for Medical Countermeasures against Radiation (CMCR) program. The CMCR program, currently in its 4th iteration, continues to be managed by the NIAID/RNCP. Although BARDA historically provided limited financial support for small Pilot Projects of interest awarded by the consortium, these contributions represent only a very small percentage of the NIAID's overall investment of >$375 M into the CMCR program from 2005 through 2022. NIAID maintains a strong and collegial trans‐government partnership with BARDA, a component of the Administration for Strategic Preparedness and Response, in the Department of Health and Human Services, but the RNCP/NIAID portfolio of grant, cooperative agreement, contract, and inter‐agency awardees is not directly tied to BARDA.RNCP REBUTTAL 2 – Education and training has continued to be a supported aspect within the CMCR program, beyond the initial 5 years of funding. For example, subsequent NIH CMCR funding opportunity announcements RFA‐AI‐19‐012“training and education materials in radiobiology research,” and urged applicants to include plans to establish“…web‐based training materials, dedicated to the scientific community, in: 1) the principles of radiobiology; 2) use of assays, methods, reagents, animal models, or technologies to study radiobiology; and 3) the regulatory process required for the licensure of new products and technologies.” In response to that mandate, the CMCR Web‐Based Radiobiology TextbookMy name is Andrea DiCarlo‐Cohen, and I lead the Radiation and Nuclear Countermeasures Program (RNCP), administered by the Division of Allergy, Immunology, and Transplantation (DAIT), National Institute of Allergy and Infectious Diseases (NIAID), within the National Institutes of Health. The RNCP provides funding to researchers to develop medical countermeasures to address gaps in diagnostics and treatments for injuries sustained during a radiological or nuclear public health emergency. I am writing to correct several inaccuracies that appeared in your December 2022 journal issue, in a paper authored by Williams JP et al.Thank you for the opportunity to address the inaccuracies made in the above‐referenced publication.


Leadership is recognized as an essential competency across healthcare and science. The LEAD (Leadership Emerging in Academic Departments) program at the Icahn School of Medicine at Mount Sinai (ISMMS) is a structured 12-month blended learning program that catalyzes personal and professional leadership skills, behaviors, and capacity.Utilizing a post-program survey design, the Leadership Program Outcome Measure (LPOM) explored self-reported impact of the LEAD program on leadership knowledge and skills in relation to personal and organizational leadership constructs. Application of leadership skills to practice was tracked via completion of a leadership-focused capstone project.Over 3 cohorts, 76 participants graduated and 50 completed the LPOM survey (68% response rate). Participants self-reported an increase in leadership skills, conveyed plans to use acquired skills in current and future leadership positions, and noted improved leadership skills across the personal and organizational domains. Comparatively less change was detected at the community level. Tracking of capstone projects found that 64% of participants were able to successfully implement their project in practice.LEAD was successful in promoting the development of personal and organizational leadership practices. The LPOM evaluation provided a valuable lens through which to assess the individual, interpersonal, and organizational impact of a multidimensional leadership training program. Leadership is increasingly recognized as core competency to advance individual and collective goals across healthcare and science, and a critical element in competency-based education for clinical and translational science (CTS) . A natioThe Icahn School of Medicine at Mount Sinai (ISMMS) has responded to this unmet need to assure leadership readiness for the CTS Junior Faculty workforce, including instructors, assistant professors and early (within the first year) associate professors, to advance leadership capacity through the design, development, implementation, evaluation, and dissemination of a Leadership Emerging in Academic Departments (LEAD) Program.We conducted a post-program online survey, administered to each cohort as they graduated from the program to assess participant’s perceived attitude and behavior change in the domains of individual leadership, organizational leadership, and community leadership. This waThe LEAD Program was designed to deliver a structured 12-month blended learning experience for junior faculty. The design of our LEAD program was initially informed by a leadership program developed at the University of Texas Southwestern Center for Translational Medicine. Our subsequent adapted program was specifically customized to our cohort of learners, using a consultative approach to learning , wherebyThe LEAD program sought to leverage diverse leadership training and professional development expertise through collaborative engagement with content experts in both the Icahn School of Medicine at Mount Sinai (ISMMS) and the Mount Sinai Health System (MSHS), including the Office of Academic Development and Enrichment (OADE) and Talent Development and Learning (TDL). To further capitalize on the wealth of institutional knowledge to advance leadership competency, program faculty were recruited from the Institute of Medical Education, the Office of Human Resources, The Center for Multi-Cultural and Community Affairs, and the Office of Diversity and Inclusion and drew upon the informed perspectives of senior leadership including the Chief Transformation Officer for the MSHS, the Deans for the medical school and scientific affairs, respectively, as well as Presidents of the Mount Sinai Hospital, Mount Sinai Queens, Mount Sinai West, and Mount Sinai Downtown. In addition, we partnered with external consultants that brought expertise not readily available within the organization, including business leaders, and leadership faculty.We designed an RFA and solicited applications from junior faculty – instructor through first-year associate professor. Applicants were asked to provide an overview of their experience in formal or informal leadership positions, their most significant achievements in clinical care, CTS research, teaching, and community engagements, as well as their reasons for wanting to participate in the LEAD program. Applicants were also asked to provide demographic information, a CV or biosketch, and a letter of support from their Department Chair, Institute or Center Director. Two program leaders reviewed applications, and applicants were scored using a rubric that ranked previous leadership experience, career stage, evidence of Department Chair, Institute or Center Director support, motivation to participate, and overall quality of the application. In addition, the program sought to gather representation from a diversity of scientific/medical disciplines, degrees, departments, and MSHS sites.The LEAD Program is built on a constructivist model , wherebyThe program is comprised of 12 interactive competency-based workshops, each focused on a specific leadership skill or attribute. To foster self-development, each participant completed the Hogan Leadership Forecast Series , the Emo–10 participants. The 60- to 90-minute meetings were facilitated by a TDL Coach and were designed to foster a safe, trusting, and confidential dialogue. This coaching opportunity offered participants a sounding board for leadership challenges, encouraged them to reflect and learn from peers, created a space for sharing lessons learned and best practices, and provided them with support in navigating the health system. It also helped them to build/expand their internal network and develop a sense of community. Almost all participants attended the Leaders’ Lab sessions, and approximately five participants per cohort took advantage of the one-to-one coaching.The LEAD workshops were supplemented by one-on-one assessment debriefs, one-to-one coaching (as requested), and an optional “Leaders’ Lab,” a monthly faculty-facilitated peer coaching session. The Hogan and EQ-i 2.0 assessment debriefs served to increase participants’ self-awareness and enabled them to establish self-development goals for the program. Upon request, the debriefs continued with one-on-one coaching to address individual development needs. The Leaders’ Lab was an optional peer-to-peer coaching that was designed to support the transfer of learning to the workplace. Through this, the participants were able to meet monthly in small groups of 6Participants were encouraged to identify a problem or issue within their own sphere of practice that could be addressed through the use of leadership concepts and newly acquired leadership skills. The final output of the program was a Leadership Capstone project, which was designed to integrate leadership principles from the curriculum in order to solve a practical issue, thereby demonstrating competence and reinforcing application of learning to practice . The aimAt the end of the program, the final capstone report was evaluated using a standardized, bespoke assessment rubric that considered the project focus, evidence of leadership impact, action planning and implementation, and outcome measures and metrics of success employed. Participants were then provided with an opportunity to make additional revisions to their written report based upon formative feedback. Lastly, participants partook in an end-of-the-year event which included a formal presentation of their project to their peers and faculty. Participants received feedback from leadership and peers during their presentation and had the opportunity to edit their projects prior to final submission. The capstone presentations were followed by a celebratory event acknowledging the accomplishments of participants and serving as a formal end to this phase of the program. An overview of the full curriculum timeline is presented in Fig. A validated post-program data collection tool, the Leadership Program Outcomes Measure tool (LPOM) , was adaThe survey uses a mixed-methods approach and includes 23 Likert-type scale questions that ask the participants to rate the perceived impact of the program as such impact relates to the programmatic goals of individual leadership , organizational leadership , community leadership, referring to the larger professional community . Each section also provided an open-ended question intended to capture respondents’ experiences in their own words and to allow for triangulation with Likert scale responses to better understand quantitative data. An additional open-ended question aimed to solicit any further feedback on the LEAD experience and/or on other dimensions of change not addressed in the questioning. The survey concluded with three 10-point scale questions asking about the importance and impact of LEAD overall.The survey was created in RedCAP, and each cohort of LEAD participants was invited to complete the survey via email at the end of their program of study. Two reminder emails spaced two weeks apart were sent to encourage participation from non-respondents. Overall response rate was 68%.Quantitative data from the LPOM were analyzed using IBM SPSS Statistics to compute means, standard deviations, and percentages. The 5-point Likert scale items were assigned numerical values  and were treated as interval data. In line with best practices, in addition to reporting mean values for each item, we also reported responses as frequencies (percentages of responses in each category) . For opeBetween 2017 and 2020, the LEAD graduated three cohorts with a total of 74 participants , new clinical programs (24%), business plans (10%), new research programs (9%), and new educational initiatives (7%). A number of important results were achieved including improvements in quality of care, patient safety, and efficiency of care processes; enhanced patient satisfaction; and new program development. Submitted capstone projects were marked using a template marking scheme, and formative feedback was provided. Fifty-two percent of the participants across all cohorts had implemented their capstone projects upon completion of their LEAD program.Participants who had not implemented their capstone projects in practice were asked on a scale of 1–10 how likely they were to do so. The mean response was 8.92. Finally, participants were asked for an overall rating of how important it was to them to implement the learning from LEAD in their practice. The mean response was 8.87.The LEAD program was perceived by participants to generate impact at multiple levels. The results of the qualitative and quantitative analysis are integrated below under the survey themes of (1) individual, (2) organizational, and (3) community levels.As result of LEAD, a majority indicated noticeable changes in individual leadership skills, attitudes, and/or behaviors related to leadership. Specifically, the most commonly endorsed individual changes include a positive effect on personal growth (4.64), increased confidence in handling leadership roles or responsibilities (3.47), increased empowerment to make a difference through leadership (4.11), and improved creative thinking (3.47). Post-completion of LEAD, participants also reported an increase in the awareness of the value of their time and connection to leadership role models (4.27).Key themes from the qualitative individual-level outcome data indicated primary changes to self-confidence and self-awareness.In terms of confidence, one participant stated: “I gained the confidence to take my goals, both research and clinical, to the next level in developing my vision into programs that could be implemented hospital-wide. I did not have the personal confidence or the leadership acumen to begin to do this prior to the LEAD program.”In terms of self-awareness, participants highlighted the value of the Hogan assessment in promoting insight into their own behavioral tendencies and motives/values/preferences. One participant stated, “I have become more self-aware. I understand my strengths and weaknesses especially pertaining to leadership style.”Related to empowerment, one participant said “I think about leadership different[ly] and now see myself as a budding leader when I didn’t before. The program helped me to think about the varied ways that I can display leadership skills and that leadership is a skill that can be developed and learned.”In terms of organization leadership, participants described how participation in the program led to wider networks of leadership contacts that transcended departmental boundaries (4.14), improved ability to network (3.58), and an ability to leverage connections to facilitate change in their own leadership practice (4.23). Participants indicated an increased ability to respond to leadership problems and situations effectively (3.89) and be more innovative in their approach to solving problems (3.7).The qualitative data from respondents described the LEAD program as a means of providing connection to organizational leadership. One participant indicated, “Since we were introduced to the hospital presidents, department chairs, and CMOs, I was better equipped to manage those conversations when I met my own hospital president to request for resource allocation.”Other graduates described the benefits of sharing perspectives with individuals from other departments or disciplines. One participant stated, “the most durable and beneficial effect of the program [is the] interaction with professionals from other departments and sites in the MSHS which offers new perspectives.”Participants also increased their understanding of MSHS as an organization and credited the LEAD program as increasing their ability to navigate the organizational landscape. As one participant stated, “I am more open to the idea of taking on a leadership role within my department; I feel better equipped to take on this kind of role because I understand the organizational structure of the hospital/medical center a lot better now.”We considered community level to be both the MSHS community and the wider medical/scientific community beyond the organization. Participants were able to increase their involvement in other areas of MSHS (3.21) but little change was reported in leadership involvement outside MSHS at the regional (2.20), national (2.23), and international levels (1.81).From the qualitative data, it appeared it was too soon for some to explore the impact of LEAD on participant’s involvement in the wider community. As one participant commented, “Although I have gained an appreciation for the importance of national and international community participation as part of the LEAD program, during the course of the program I have not yet engaged in these efforts. However, I have made it a personal goal to do so in the upcoming year.”A smaller number of participants were able to provide concrete examples of wider community involvement. One participant shared “1. I became treasurer of a medical society 2. I became involved in organizing two international scientific conferences. 3. I became an associate scientific advisor for a major journal. 4. I was selected as reviewer for my first NIH study section.”–3 years post) of capstone projects found that 64% of participants were able to successfully implement their project in practice.The LEAD was created, implemented, and evaluated to advance competency-based leadership capacity among junior faculty clinical and translational science investigators. The successful attainment of leadership competencies was evaluated both subjectively, the validated and adopted LPOM tool, designed to assess the self-reported impact of this training on leadership capability by participants, and objectively via the successful completion of a rubric-assessed capstone project. Utilizing the LPOM, participants reported an increase in self-assessed leadership skills, conveyed plans to use acquired skills in current and future leadership positions, and noted improved leadership skills across the personal and domains. In addition, participants demonstrated effective integration of leadership concepts and through the development and successful implementation of a wide range of leadership capstone projects. Subsequent tracking , and knowledge of the MSHS organizational structure. Participants largely attributed these changes to their facilitated interactions with senior leadership at Mount Sinai, for example, hospital presidents and chairs. Participants engaged in facilitated Q&A sessions with these leaders, which provided the opportunity for them to make new connections, practice their communication skills with senior leaders, and gain a better understanding of the extremely complex matrix organizational structure at MSHS. We also have seen the development of a strong support network between participants, particularly through the Leaders’ Lab, resulting in a community of practice that has extended beyond the program.Comparatively less change was detected at the community level. It may be that change in this domain takes longer to implement and may be more challenging as it is linked with a shift in organizational culture to further advance the cross-collaboration that requires a breakdown of established silos.The existing organizational culture, with its established systems and processes, may impact participants’ ability to implement their capstone project. Although a small majority of the LEAD cohort were able to implement their capstone projects during the LEAD program (one participant was awarded a FOJP Innovation Grant for his capstone work), the remaining participants did not implement their projects although they showed a strong desire to do so following completion of the LEAD program. We hypothesize that time and workload constraints may be another barrier to successful project implementation during the LEAD program.To address the lack of change at the community level, we recognize a need to support LEAD participants in their efforts to extend their influence to the wider professional community both within and beyond MSHS. Evidence suggests that, in general, the culture of an organization affects the degree to which learned behavior will be transferred to practice . ParticiIn discussing the importance (and benefit) of measuring leadership change at the individual, organizational, and community levels, we also recognize the limitations of using self-reported data as a substitute for more objective observational data. Participants may provide an underestimation or overestimation of the knowledge and skills gained with the latter being more likely because of the potential for social desirability bias and effort justification bias. We also must highlight the difficulty in accessing observational data in this context. Furthermore, only focusing on these participant-level, self-reported outcomes neglects the impact these leaders have on their subordinates, peers, and superiors .The LPOM provided us with an in-depth self-assessment of change following the LEAD program. In the future, we intend to also track objective measures of productivity, including promotions, new leadership roles, and publications based on capstone projects. In addition, emerging data suggest that leadership training contributes to resilience and we intend to more rigorously assess this contribution in future iterations of the program. Future plans for the LEAD program involve including a 360-degree assessment process, allowing participants to gather data from peers and colleagues to provide an external perspective of change. Furthermore, the launch of the Ambassador program provides a further opportunity to advance leadership capacity though continued learning with peers.LPOM has provided a lens through which to assess the individual, organizational, and community impact of a multidimensional leadership training program. LEAD provides an educational initiative that promotes the development of personal and organizational leadership practices. This is important, given the overwhelming need for leadership skills to promote early career faculty in meeting the demands of advancing science to meet the complex demands of translational science. We have presented findings from three cohorts of participants; future research will evaluate the longer-term impact of the LEAD program, especially with regard to the development of an alumni program and an ongoing approach to the leadership development at the MSHS.