Study On The Novel Predictive Model And Biomarkers For The Differential Diagnosis Of Pulmonary Nodules Based On Multi-Level Data

The latest national cancer report released by the National Cancer Center shows that lung cancer ranks first in the number of new cases and deaths of malignances in China."Early detection,early diagnosis and early treatment" is the key to improving the survival rate.The promotion of chest low-dose CT(LDCT)screening has effectively reduced the mortality of lung cancer,but the concomitant problem is the significant improvement of the clinical detection of pulmonary nodules.Pulmonary nodules refer to high-density shadows with a diameter of ≤3cm found by CT scan.According to their density,they can be divided into ground glass nodules(GGN),subsolid nodules(SSN)and solid nodules(SN).Their pathological causes are complex,which may be benign diseases such as infection,granuloma and bleeding,and also can be malignant tumors.In 2002,the National Cancer Institute reported that the detection probability of pulmonary nodules was 24.2%in 53,454 individuals with high-risk of lung cancer,and the proportion of benign nodules was as high as 96.4%.Accurate diagnosis of pulmonary nodules has become an important clinical problem we confront.However,there remains problems such as difficulty in differential diagnosis between benign and malignant lesions,lack of clear diagnosis and intervention targets,unclear progression mechanism and so on,resulting in huge social anxiety and potential misdiagnosis and possibility of over diagnosis and over treatment.Accordingly,we conducted a total of four parts of research.In the first part,the clinicopathological features of hospitalized patients with pulmonary nodules in our hospital in recent 5 years were retrospectively analyzed.The clinicopathological features of large-scale pulmonary nodules in Henan were reported for the first time.It was found that the proportion of patients with pulmonary nodules increased year by year in recent 5 years,in which solitary pulmonary nodules accounted for the majority of patients and were associated with higher malignant risk;In the second part,for the differential diagnosis of benign and malignant solitary pulmonary nodules,the classical Mayo model was externally verified and corrected by the clinical and imaging data from three large tertiary hospitals.Then,the first differential diagnosis model of benign and malignant solitary pulmonary nodules based on population data in Henan Province was constructed,internal and external verification was carried out;In the third part,the high affinity CD 146 monoclonal antibody was developed and used to detecte level of CD 146 in the peripheral blood of patients with pulmonary nodules as a new diagnostic marker,which was further incorporating into the diagnostic model of solitary pulmonary nodules established in the second part;In the fourth part,the single-cell transcriptome sequencing combined with single-cell TCR/BCR sequencing was used in normal lung tissue,adenocarcinoma in situ(AIS)and early invasive lung adenocarcinoma(IAC).The cellular and molecular characteristics of various cell subsets in the immune microenvironment at different pathological stages were characterized and compared,so as to further explore the potential diagnostic and therapeutic targets.In addition,the high expression of differential genes in macrophage subsets found in single-cell sequencing data was verified in macrophages in alveolar lavage fluid of patients with pulmonary nodules,suggesting the potential of detecting macrophages in alveolar lavage fluid under accurate navigation for the differential diagnosis of benign and malignant pulmonary nodules,in order to provide new ideas for the early differential diagnosis of pulmonary nodules and lung cancer.Part Ⅰ:5-Year Single Center Retrospective Study of Clinicopathological Features of Patients with PulmonaryNodulesObjectiveTo understand the clinicopathological features,disease spectrum and related factors of benign and malignant pulmonary nodules in our center in recent 5 years.MethodsFrom January 2017 to December 2021,the inpatients diagnosed with"pulmonary nodule","pulmonary nodule","lung mass","lung mass","lung space occupying","lung cancer","lung malignant mass","lung malignant tumor","lung shadow","lung high-density shadow" and "lung high-density shadow" were collected retrospectively.Cases with the maximum diameter of pulmonary nodules less than 3cm were selected by imaging data,and the clinical data were further collected and analyzed.ResultsFrom 2017 to 2021,the proportion of patients with pulmonary nodules in relevant departments increased year by year(Z=13140.9,P<0.001).The average age of all patients with pulmonary nodules was 59.76±12.3 years old(14 years old-91 years old).Women accounted for 50.9%(3113/6115)of all the patients.87.7%(5363/6115)were confirmed by pathological test,and most of them were confirmed by surgery(4042/6115,66.1%).Most of the lesions were solid nodules(3272/6115,53.5%),and the rest included part solid GGN(1993/6115,32.6%)and pure GGN(856/6115,14.0%).Most of them were solitary pulmonary nodules(4372/6115,71.5%).The most common benign pulmonary nodules were nonspecific inflammatory lesions(22.8%,337/1480),followed by tuberculosis(21.1%,312/1480),granulomatous diseases(13.2%,195/1480),hamartoma(9.7%,144/1480),invasive adenocarcinoma(70.2%,2726/3883),squamous cell carcinoma(9.5%,369/3883),small cell lung cancer(5.2%,202/3883)accounted for most of the malignant lesions.Older age(P<0.001),sub-solid nodules(P<0.001),solitary nodules(P<0.001),smoking history(P<0.001)and cancer history(P<0.001)were associated with the risk of malignancy.Most(62.3%)patients with malignant pulmonary nodules were in stage I at the time of diagnosis,however 14.6%of patients with malignant pulmonary nodules had metastasis at the time of diagnosis.ConclusionThe data of our center showed that the proportion of patients with pulmonary nodules has increased year by year in the past five years.Invasive adenocarcinoma and nonspecific inflammatory diseases are the most common pathological types of malignant pulmonary nodules and benign pulmonary nodules respectively.In addition,malignant nodules are mostly sub-solid and solitary pulmonary nodules,which are related to smoking,older age,and previous cancer history.Part Ⅱ:Establishment and Verification of Multifactor Model for the Differential Diagnosis of Benign and Malignant Solitary Pulmonary NodulesObjectiveFor the differential diagnosis of benign and malignant solitary pulmonary nodules,the Mayo model was externally verified and corrected,and a new diagnostic model of solitary pulmonary nodules was established based on the multi-center data in Henan Province.Methods1450 patients with solitary pulmonary nodules who underwent surgery in three tertiary hospitals in Henan Province were included,and were divided into training set,internal verification set and external verification set(n=849,365,236).The Mayo model was externally verified and re-corrected with the data of the training group,and a new Logistic regression model was established.The area under curve(AUC),accuracy,sensitivity,specificity,positive predictive value(PPV)and negative predictive value(NPV)of receiver operating characteristic curve(ROC)were used to evaluate the overall performance of each model.Finally,the new model is verified in the external validation data set.The AUC values between different models were compared by Delong test,and the difference was statistically significant(P<0.05).ResultsThe AUC value of Mayo model in the training set is 0.653(95%CI:0.613-0.694).After recalculating the coefficients of all covariates in the original Mayo model,the original model was corrected,and the AUC value of the corrected model was 0.671(95%CI:0.635-0.706).Then we constructed a new model.The AUC of the new model in the training set was 0.891(95%CI:0.865-0.917),the accuracy was 83.86%,the sensitivity was 85.1%,the PPV was 92.59%,and the NPV was 65.06%.In the internal validation dataset,the accuracy was 88.22%,and the sensitivity was 92.10%;Specificity was 72.97%,PPV was 93.06%,NPV was 70.13%.In the external validation set,the accuracy was 81.78%,sensitivity was 79.65%,specificity was 83.74%,PPV was 81.82%,NPV was 81.75%.The AUC of the new model in the internal validation set was 0.888(95%CI:0.842-0.934),which was significantly higher than that of the corrected Mayo model(AUC=0.577,95%CI:0.509-0.646,P<0.001)and the original Mayo model(AUC=0.609,95%CI:0.5440.675,P<0.001).In the external validation set,the AUC of the new model was 0.876(95%CI:0.831-0.920),which was higher than that of the corrected Mayo model(AUC=0.706,95%CI:0.640-0.772,P<0.001)and the original Mayo model(AUC=0.705,95%CI:0.639-0.772,P<0.001).ConclusionThe first differential diagnosis model of benign and malignant solitary pulmonary nodules based on large-scale population data in Henan Province is established.The new model can more accurately distinguish the benign and malignant of pulmonary nodules than the classical Mayo model,which is expected to assist in the differential diagnosis of benign and malignant pulmonary nodules in clinic.Part Ⅲ:Further Optimization of the Differential Diagnosis Model of Solitary Pulmonary NodulesObjectiveHigh affinity CD 146 monoclonal antibody was developed by phage library display technology for the detection of clinical samples,and the level of serum CD 146 was incorporated into the diagnostic model of solitary pulmonary nodules,to further improve the diagnostic efficiency.MethodsRelying on the natural phage antibody display library constructed by the research group previously,four rounds of screening of CD 146 monoclonal antibody were carried out,and its affinity was detected by ELISA and flow cytometry.Serum samples from patients with pulmonary nodules were collected for detection.CD 146 level was incorporated into the clinical prediction model,and its optimization ability for the clinical prediction model was evaluated by AUC.ResultsA total of 5 high monoclonal antibodies(MM01-MM05)were obtained,and MM01 has the highest affinity.WB,immunohistochemistry,fluorescence staining and flow cytometry showed that MM01 had good affinity and could detect the expression of CD146 in cells and tissues.A total of 126 newly diagnosed patients with pulmonary nodules were enrolled,with a median age of 61.4±12.5 years(34y-79y),49.2%were male(62/91)and 32.5%were smokers(41/91).The level of serum CD146 in patients with malignant nodules was 235.0±52.8ng/ml(94.2 ng/ml-405.0 ng/ml),which was higher than 201.7±41.8 ng/ml(132.9 ng/ml-294.2 ng/ml)in patients with benign pulmonary nodules(P=0.0014);Adenocarcinoma 229.0±50.443 ng/ml(94.2 ng/ml-352.9 ng/ml),squamous cell carcinoma 262.2±47.87(165.4 ng/ml-405.0 ng/ml),which were higher than those in patients with benign pulmonary nodules(P=0.0234,P=0.0003);There was no significant difference between patients with benign lung cancer and patients with small lung nodules(P=0.1639).Age(P=0.6883),smoking history(P=0.3107)and gender(P=0.8544)were not related to the level of serum CD146.The AUC value of CD146 serum level in the differential diagnosis of benign and malignant pulmonary nodules was 0.695(95%CI:0.598-0.791,P=0.001),which was higher than CEA(95%CI:-0.0287-0.212,Z=1.492,P=0.1356),NSE(95%CI:-0.0226-0.247,Z=1.632,P=0.1028),SCC(95%CI:-0.0386-0.237,Z=1.410,P=0.1583),CYFRA21-1(95%CI:-0.00365-0.0451,Z=1.667,P=0.0956),but there was not statistically significant.Fitting the diagnostic model of solitary pulmonary nodule established in the previous part to this part of the population,the AUC value is 0.740(95%CI:0.651-0.829,P=0.000).After combining the serum CD 146 level with the prediction model,the diagnostic AUC is 0.760(95%CI:0.671-0.850,P<0.001),and the increase of AUC was 0.0209(95%CI:0.045-0.0869).The likelihood ratio test shows that the increment was statistically significant(P<0.001),The AUC was 0.788(95%CI:0.705-0.872,P<0.001)after incorporated CD 146 and other lung cancer biomarkers and the increment was 0.0481(P<0.001).ConclusionCD 146 is expected to become a new differential diagnostic marker for benign and malignant pulmonary nodules.The combination of the prediction model with the level of CD 146 in peripheral blood and other biomarkers is expected to further improve its ability in the differential diagnosis of benign and malignant pulmonary nodules.Part Ⅳ:Analysis of Cellular and Molecular Characteristics of Immune Microenvironment of Ground Glass Pulmonary Nodules and the Study of Potential Diagnostic and Therapeutic TargetsObjectiveThe most common diagnosis of malignant ground glass pulmonary nodules is atypical adenomatous hyperplasia(AAH),AIS,micro-invasive adenocarcinoma(MIA)and IAC.It has become a dilemma in clinical diagnosis due to the characteristics of low metabolism and inert growth.The lack of understanding of the characteristics of its immune microenvironment hinders the exploration of new diagnosis and treatment targets.This part intends to understand the cellular and molecular characteristics of immune microenvironment at different stages of early lung adenocarcinoma with ground glass pulmonary nodules through single-cell transcriptome and TCR/BCR sequencing,so as to provide theoretical basis for the discovery of new diagnostic and therapeutic targets.MethodsThe surgical specimens of patients with pulmonary nodules were collected,the single cells were sorted,and the library was built through the platform of 10X genomic company,and the second-generation transcriptome and TCR/BCR were sequenced.The subsequent bioinformatics analysis was carried out by Seurat package etc.The changes of lipid droplets in cells were observed by multicolor fluorescence.The expression of macrophage marker genes in alveolar lavage fluid of patients with pulmonary nodules was verified by immunohistochemistry,real-time quantitative polymerase chain reaction(RT-qPCR)and Western-Blot(WB).ResultsA total of 7 patients,aged 34-65 years,were included,including 4 males and 2 smokers.A total of 12 samples were obtained for sequencing,including 3 cases of AIS,4 cases of early IAC with ground glass nodules and 5 cases of paired normal control lung tissue(nlung).A total of 38814 single cells were obtained after quality control.nLung,AIS and IAC have different immune microenvironment characteristics.In different stages of nlung-AIS-IAC,the metabolic synthesis,intercellular signal transduction,stress response and other related pathways of tumor cells were enriched,and the expression of intercellular connectivity and adhesion related pathways decreased;Cillium structure and function and mitochondrial function related pathways were significantly enriched in AIS and decreased significantly in IAC stage;In the AIS and IAC stages,regulatory CD4+T cells were enriched,NK cytotoxicity weakened and the number decreased,but there were no depleted CD8+T cells.At the same time,T cells showed the expansion of new clonal types and the transformation of main clonal cell subsets;B cells were significantly enriched in AIS and IAC stages,and new clonal expansion appeared;There is a significant enrichment of monocyte derived macrophages in AIS and IAC,which highly expresses the gene of tumor associated macrophage(TAM)and enriches a variety of cancer-related pathways.With the changes of multiple lipid metabolism pathways,it further proves the accumulation of macrophage lipid droplets in the microenvironment of early lung adenocarcinoma by polychromatic immunofluorescence.RT-qPCR and WB results showed that the high expression of related genes in single cell sequencing data could be detected by alveolar lavage fluid macrophages in patients with lung adenocarcinoma.ConclusionIn this part,the single-cell transcription atlas of nlung-AIS-IAC was created,and the cellular and molecular characteristics of immune microenvironment in different stages of lesions were analyzed and compared,which provided some theoretical basis for the discovery of new diagnostic and therapeutic targets in the future.Detecting the changes of macrophages in alveolar lavage fluid of patients with pulmonary nodules provides a new idea for the early diagnosis of pulmonary nodules in the future.