Construction Of A Risk Prediction Model For Venousthromboembolism In Patients With Lungcancer Based On Multi-dimensional Indicators And Research On The Mechanism Of LncRNA MALAT Regulating NRF2 In Pulmonary Vascular Remodeling In CTEPH Rats

Objective:（1）The aim of this single-centre 10-year retrospective observational study was to evaluate the profile of Chinese cancerous patientscomplicated with venousthromboembolism（VTE）based on demographic features,clinical characteristics,and medication exposure.（2）To systematically review the incidence of VTE in patients with lung cancerby different driver gene mutations,and to further explore the associations of driver gene mutation status with with the risk of VTE.It is expected to screen VTE high-risk populations based on driver gene mutation status.This suggests that incorporating them into a risk assessment model may improve performance.（3）To develop and validate a specific risk-stratificationnomogram model for the prediction of VTE inhospitalized patients with lung cancer using readily obtainable demographic,clinical and therapeutic characteristics,thus guiding the individualizeddecision-making on thromboprophylaxis on the basis of VTE risk levels.（4）To determine the differential expression of LncRNA MALAT1 and NRF2 in CTEPH rat model and to verify that MALAT1regulates NRF2 and promotes pulmonary vascular remodeling through animal experiments.Methods:（1）Consecutive 1482 patients with solid tumor complicated with VTE at a tertiary center between 2012 and 2021 were retrospectively enrolled.Data were collected on demographics,comorbidities,discharge diagnoses,laboratory examination data,treatment details,and imaging description of the lesion.（2）A complete search of PubMed,EMbase,Web of Science,Cochrane Library,CNKI,Wanfang and VIP Database were performed to obtain eligible studies on the the correlation between driver gene utation status and vehicle crash risk of VTE published to 3rd July 2023.During the review process,two independent reviewers assessed the methodology,extracted data,and screened the literature independently.（3）We performed a retrospective case-control study among newly diagnosed lung cancer patients hospitalized between January 2016 and December 2021.Included in the cohort were 234 patients who developed VTE and 936non-VTE patients.The patients were randomly divided into the derivation group（70%,165 VTE patients and 654 non-VTE patients）and the validation group（30%,69 VTE patients and 282 non-VTE patients）.Cut off values were established using a Youden′s Index.Univariate and multivariate regression analyses were used to determine independent risk factors associated with VTE.Variance Inflation Factor（VIF）was used for collinearity diagnosis of the covariates in the model.The model was validated by the consistency index（C-index）,receiver operating characteristic curves（ROC）and the calibration plot with the Hosmer-Lemeshow goodness-of-fit test.The clinical utility of the model was assessed through decision curve analysis（DCA）.Further,the comparison of nomogram model with current models（Khorana,Caprini,Padua and COMPASS-CAT）was performed by comparing ROC curves using the De Long’s test.（4）We established a rat model of CTEPH by repeated autologous thromboembolization,a variety of assays were carried out two and four weeks after the initial injection.The primary outcome was pulmonary haemodynamics,as assessed by measurement of the right ventricular systolic pressure（RVSP）,mean pulmonary arterial pressure（m PAP）,and hemodynamic parameters examined by color Doppler ultrasound.HE and Masson staining were conducted to detect the degree of pathological damage and fibrosis in rat lung tissue,respectively.HE staining and Masson staining reveal thickening and fibrosis in the pulmonary arteries of CTEPH rats.Rats were randomized to receive either adeno-associated virus expressing MALAT1/NRF2 or an adeno-associated virus control at1.3×10¹²v.g/ml four weeks after modeling.Rats were randomly divided into the Ctrl,CTEPH,CTEPH+sh-NC,CTEPH+sh-MALAT1,CTEPH+oe-NRF2,CTEPH+sh-MALAT1+sh-NRF2 groups.The expression levels ofα-SMA and NRF2 after modeling were determined by western blotting and immunofluorescence staining.RT-qPCR was used to detect the level of plasma and tissue MALAT1 in all groups.Results:（1）The overall incidence of clinical VTE was 1.35%in hospitalized patients with cancer in our center.Lung cancer was the most frequent tumor subtype for developing VTE events,accounting for 24.83%of all cases.Over half of the patients（66.60%）were observed to have an increased risk of VTE within the first 6 months of cancerdiagnosis.Close to half of the patients（46.49%）had received chemotherapy within6 months prior to the diagnosis of VTE.（2）Thirty-two studies involving 18,032 cases were obtained for Meta-analysis.This meta-analysis revealed that the overall incidence of VTE in NSCLC patients harboring mutations in driver genes was 19%[95%CI（15%,23%）].Incidence of VTE in ROS1 gene fusions group[95%CI（22%,39%）]were significantly higher than that in EGFR/BRAF gene mutation group（EGFR:16%[95%CI（12%,20%）];BRAF:14%[95%CI（3%,24%）]）.Besides,the presence of ALK/ROS1gene fusions and positive PD-L1 expression are related to a significant increased risk for development of VTE[ALK:OR=2.28,95%CI（1.89,2.76）,P<0.001;ROS1:OR=3.18,95%CI（1.89,5.35）,P<0.001;PD-L1:OR=1.85,95%CI（0.75,1.80）,P=0.002].However,the pooled ORs indicated that EGFR/BRAF gene mutation were not significantly associated with risk of VTE in overall analysis[EGFR:OR=1.16,95%CI（0.75,1.80）,P=0.495;KRAS:OR=1.54,95%CI（0.98,2.43）,P=0.061].Sensitivity analysis and Begg’s test and Egger’s test confirmed the stability and reliability of the meta-analysis.（3）The predictive nomogram modle comprised eleven variables:overweight（24-28）defined by body mass index（BMI）:[odds ratio（OR）:1.90,95%confidence interval（CI）:1.19-3.07],adenocarcinoma（OR:3.00,95%CI:1.88-4.87）,stage III-IV（OR:2.75,95%CI:1.58-4.96）,Central venous catheters（CVCs）（OR:4.64,95%CI:2.86-7.62）,D-dimer levels≥2.06mg/L（OR:5.58,95%CI:3.54-8.94）,PT levels≥11.45sec（OR:2.15,95%CI:1.32-3.54）,Fbg levels≥3.33g/L（OR:1.76,95%CI:1.12-2.78）,TG levels≥1.37mmol/L（OR:1.88,95%CI:1.19-2.99）,ROS1 rearrangement（OR:2.87,95%CI:1.74-4.75）,chemotherapy history（OR:1.66,95%CI:1.01-2.70）and radiotherapy history（OR:1.96,95%CI:1.17-3.29）.Collinearity analysis with demonstrated no collinearity among the variables.The resulting model showed good predictive performance in the derivation group（AUC 0.865,95%CI:0.832-0.897）and in the validation group（AUC 0.904,95%CI:0.869-0.939）.The calibration curve and decision curve analysis（DCA）demonstrated that the risk stratification nomogram exhibited strong consistency and clinical utility.Futher,the area under the ROC curve for the specific VTE risk-stratification nomogram model（0.904;95%CI:0.869-0.939）was significantly higher than those of the KRS,Caprini,Padua and COMPASS-CAT models（Z=12.087,11.851,9.442,5.340,all P<0.001,respectively）.（4）The hemodynamic findings showed that the RVFW、RVID_dand m PAP of CTEPH rats increased markedly,but PAT/PET ratios and TAPSE values significantly decreased（P<0.05）.Pathology revealed increased the vascular wall thickness（WT%and WA/TA）in CTEPH rats compared with control rats（P<0.001）.Immunofluorescence staining showed that the fluorescence intensities ofα-SMA in CTEPH rats were elevated,while the fluorescence intensities of NRF2 were decreased（P<0.05）.m PAP and RVSP were significantly lower in CTEPH+sh-MALAT1 group and CTEPH+oe-NRF2 group,when compared with CTEPH group and CTEPH+sh-MALAT1+sh-NRF2 group,respectively.According to He and Masson staining,silencing MALAT1 and overexpressing NRF2significantly ameliorated the severity of pulmonary vascular remodeling in rats.There was no statistically significant difference in all the evaluated indicators between the CTEPH+sh-NC and CTEPH groups（P>0.05）.Conclusions:（1）Patients with lung cancer were considered at high risk for VTE.The assessment and monitoring of VTE in patients with cancer within the first 6 months of cancer diagnosis should be strengthened.VTE occurrence was closely related to advanced age and stage,adenocarcinoma,obesity and noval anticancer therapies in patients with cancer.（2）Significant differences in the incidence of VTE in NSCLC patients harboring distinct mutations in driver genes.the presence of ALK/ROS1 gene fusions and positive PD-L1 expression were associated with increased risk for development of VTE,with no significant associations with EGFR/BRAF gene mutation.Therefore,detecting driving gene mutations for lung cancer patients would be valuable for Early screening for the VTE high-risk population.（3）A high-performance nomogram model incorporated available clinical parameters,genetic and therapeutic factors was established,which can accurately predict the risk of VTE in hospitalized patients with lung cancer and to guide individualized decision-making on thromboprophylaxis.Notably,the novel nomogram model was significantly more effective than the existing well accepted models in routine clinical practice in stratifying the risk of VTE in those patients.Future studies from multiple clinical centers are required for external validation.（4）The development of pulmonary artery hypertension and thickening in the pulmonary artery is observed in the CTEPH rat model.MALAT1 was high-expressed while NRF2 was low-expressed in CTEPH rat model.silencing MALAT1and overexpressing NRF2 significantly ameliorated the severity of pulmonary vascular remodeling in rats.