Natural Growth Evaluation And Detection Of Influence Factors For Pulmonary Nodule With Ground-glass Opacity

Lung cancer is the leading cause of cancer death in men and the second leading cause of death in women worldwide.In recent years,with the low-dose spiral CT has been widely used,more and more pulmonary nodules detected.However,persistent pulmonary nodules with ground glass opacity（GGO）mostly were malignant tumor,most of which represent pre-invasive adenocarcinoma.If these early stage adenocarcinoma were timely diagnosed and treated,which significantly reduced lung cancer-related mortality in the population.For the screened pulmonary nodules with GGO,from the pathological point of view,From the atypical adenomatous hyperplasia（AAH）,adenocar-cinoma in situ（AIS）,minimally invasive adenocarcinoma（MIA）to invasive adenocarcinoma（IAD）,the components of solid increase such as pure GGO,mixed-GGO,sub-solid nodules and solid nodule.If the solid component of pulmonary ground glass increases and the degree of pathological progress,then the risk of blood metastasis,invasion of pleura or lymphatic metastasis develop.However,for pulmonary nodules,the current guidelines suggest the annual follow-up or long-term observation.Some current studies,including chemotherapy,immunotherapy,targeted therapy and other attempts,have poor effects on GGOs,and no unified treatment recommendations have been made.The root cause may be that the blood supply of glassed nodules is small,and drugs are difficult to reach the lesion,resulting in efficacy.Moreover,due to the small sample size of lung nodules,it is difficult to form a model of lung carcinoma in situ,so the treatment of lung nodules has been in the exploratory stage.From Fisher guidelines（2017）to NCCN guidelines（2022）,most of them were divided into pure GGO,partial solid GGO and solid pulmonary nodules according to the solid component.Different follow-up strategies for pulmo-nary nodules with GGO,the most critical factors whether surgical procedure should be conducted were nodule size,increasing of solid components or the size of nodule.However,the guidelines did not mention whether the imaging features of relevant nodules,such as lobulation sign,pleural indentation and vacuole,were related to the growth of nodules.The maximum diameter of the growth of nodules did not reflect the authenticity of the growth.Recent studies suggested that the growth of nodules should be based on the volume,density and finally the quality of nodules.At present,few studies have shown which indicators can predict nodule growth or pathological progression.Therefore,in this study,the pulmonary nodules with ground glass were observed for long-time follow-up,finally confirmed by pathology.In clinical studies,the imaging characteristics of pulmonary nodules were retrospectively analyzed,and the natural growth rules of nodules with increasing growth were analyzed,and whether there was a relationship between a certain index and growth was analyzed through hematological characteristics.In basic research,factors related to the progression of early lung adenocarcinoma were analyzed from the characteristics of pathological immunochemistry and immune microen-vironment,hoping that the research results could more accurately guide the clinical follow-up strategy of patients with lung nodules,and the results of basic research could provide direction for the treatment of lung ground-glass nodules.Part One The natural growth rate of pulmonary nodules with ground-glass opacity and association of clinic features with itObjective:To investigate the growth rate of pulmonary nodules with ground-glass opacity by volume（VDT）and mass doubling time（MDT）and explore the high risk factors of clinical-pathological features with it.Methods:We collected 116 cases with persistent pulmonary nodules,including 62 patients as stable vs 54 patients in growth group,from 2015 to2021.The clinical features of the patients included age,sex,smoking history,family history of tumor,postoperative pathology,degree of infiltration and solid compnent of pulmonary nodules.Pathological diagnoses of GGNs after surgically resected were also recorded.Then the cases were divided into two groups,i.e.,stable and growing groups.The duration of follow-up and the variation in GGOs volume and mass were recorded.The morphologic changes and three-dimensional segmentation of GGO nodules were used to calculate the VDT and MDT based on a modified Schwartz model.Then,independent sample t-test was used to investigate the relationship between growth and their features,three statistical methods of logistic regression model analysis were used to explore the potential risk factors of malignant pulmonary nodules with GGO by SPSS23.0.Results:1.In 116 patients,including 62 patients as stable vs 54 patients in growth group.Lobulation sign,spiculation sign,pleural indentation,air bron-chogram,vessel convergence,vacuole sign were observed in 45（38.8%）,22（18.9%）,24（20.7%）,10（8.6%）,59（50.8%）,and 41（35.3%）,respectively.Compared with the stable group,air bronchogram（16.7%vs 1.6%,P=0.006）,vessel convergence sign（70.4%vs 33.9%,P<0.001）,vacuole sign（48.1%vs 24.2%,P=0.011）,and the median vessel diameter entering into the GGN（1.25 vs 0,P<0.001）were significant different.In a multivariable analysis,the patient age（P=0.037）,the solid component of GGN（P<0.001）,the vacuole sign（P=0.002）,and the vessel diameter through pulmonary nodules（P=0.013）were closely related to the growth of persistent malignant pulmonary nodules.2.Because the solid component was the main risk factor for growth,the analysis of the subgroup was necessary for the GGO with a solid component ratio<0.5.Multivariable analysis showed the vacuole sign（P=0.019）,and the vessel diameter through pulmonary nodules（P=0.027）were the risk factors for the growth.3.Cut-off value of age was 54 with its specificity 88.9%and sensitivity54.8%,and cut-off value of tumor blood vessel diameter was 0.9 mm with its specificity 82.3%and sensitivity 66.7%.While in subgroup analysis,for the GGO CTR<0.5（C（the maximum diameter of consolidation in tumor）/T（the maximum diameter of the whole tumor including GGO）ratio）,tumor blood vessel diameter（P=0.027）was important during the growing processes of nodules4.The averaged VDT and MDT of the growth GGOs were 973 days and671 days,respectively.MDT varied significantly different among the three groups of components（CTR）<0.25:799.92days,0.25≤CTR≤0.5:483.7,and CTR>0.5:442.8 days)（P<0.05）.The growth rate of GGOs with CT imaging vacuole sign may be slower than those without this feature（696.58-days vs428.25days P<0.05）.Conclusions:The age,solid component of GGNs,blood vessel diameter entering into GGN lesion and vacuole sign were closely associated with the growth rate of GGNs.Blood vessel diameter entering into GGO lesion and vacuole sign maybe the high risk factors for the growth of GGNs.The results of this study would provide evidence for effective strategies recommendation.Part Two The correlation research between peripheral blood index and the growth of early stage malignant pulmonary ground-glass opacityObjective:To detect the relationship between peripheral blood index and the growth of pulmonary ground-glass opacity（GGO）in very early lung adenocarcinoma.Methods:All patients with GGO underwent surgical procedures were enrolled,including the time of follow-up and the variation tendency of GGO.The value of lymphocytes,platelets,NLR,CEA MONO and PLR was divided from the peripheral blood.Age,gender,smoking history,histology,tumor size,and stage were the factors examined in this study.At the same time,relevant indicators of the proportion of immune cells in patients’lymphocytes were collected.Logistic regression was used to evaluate the value of NLR and the cut-off value was calculated by SPSS22.0.Results:1.In the whole cohort,30 cases of growing GGO and 43 cases of stable GGO underwent surgical procedures were diagnosed as lung adenocarcinoma.the mean counts of white cell,neutrophil and NLR in growing GGO group were different from the stable GGO group（P=0.031,P=0.002,P<0.001）.However,there was no significant difference in mean value of lymphocytes,platelets,NLR,CEA MONO and PLR.Through the analyses of logistic regression between two group,we found that age（P=0.020）and NLR（P=0.001）were significant associated with the growing GGOs in the univa-riate analysis.2.Multivariate analysis showed that NLR predicted the growth of GGOs（odds ratio 5.198,95%CI 1.583-14.581,P=0.002）.Reciever operating charac-teristics analysis for NLR showed the optimal cut-off value of 2.38,with a sensitivity of 60.0%and specificity of 81.4%.NLR appeared to have the value as a promising clinical predictor of the growth of GGN.3.In order to exclude the influence of persistent benign nodules on the value of NLR,persistent benign pulmonary nodules confirmed by pathology were collected in this study for comparison,and it was found that there was no statistical significance in the difference of NLR between benign and malig-nant.The inflammatory lesion group included tuberculosis,structural pneu-monia and epithelioid granulomatous lesions（a total of 18 cases）.The mean value of NLR in the inflammatory lesion group（2.41±1.32）was not higher than that in the malignant nodules group（2.35±1.15）,and the differ-ence was not statistically significant（P=0.851）.4.By subgroup analysis of the proportion of immune cells in lympho-cytes,Logistic regression,multivariate analysis results showed that immuno-regulatory T cells（CD4+CD25+CD127dim）were the risk factor for the growth of pulmonary ground glass nodules（P=0.009,OR:0.471,95%CI:0.268-0.828）,indicating that the decrease of immunoregulatory T cells in lymphocytes may be closely related to the growth of early malignant pulmo-nary grinding glass nodules.Conclusions:Our study demonstrated the NLR appeared to have the value as a promising clinical predictor of the growth of GGOs.Subgroup analysis of the proportion of lymphocyte classification suggests that immune regulation T cells may be associated with the growth of malignant GGNs,hoping that they can be used as markers in the future to provide help in screening lung nodules.Part Three Growth-related immunoprotein expression in the tumor microenvironment of early stage malignant ground-glass nodules of the lungObjective:This study intends to explore the factors affecting the growth of pulmonary nodules in the natural process by immuno-histochemical method..Methods:The expressions of cyclooxygenase-2（COX-2）,Ki67,vascular endothelial growth factor（VEGF）,CD44V6,epidermal growth factor receptor（EGFR）,double microsome 2（MDM2）and transforming growth factor（TGF）-β1 in pulmonary nodules were detected by immunohistochemical method so as to explore the relationship between immunoprotein expression and the growth of GGN by SPSS22.0.Results:1.40 cases of pulmonary nodules followed up for more than 3 years were divided into growth group（n=20）and stable group（n=20）.Compared with stable pulmonary nodules,the positive rates of COX-2,Ki67 and VEGF in the growth group were 85%,80%and 55%,respectively.There was significant difference between the stable group and the growth group（P<0.05）.The correlation between other indexes and the growth of pulmonary nodules was not statistically significant(P_cd44v6=0.104;P_EGFR=0.337;P _MDM2=0.49;P _TGF-β1=0.141).2.In the subgroup of patients with non-invasive lung cancer,there was a correlation between VEGF and the growth of pulmonary nodules（P<0.05）.Conclusions:The high expression of COX-2,Ki67 and VEGF proteins may be significantly related to the growth of pulmonary nodules,and VEGF may be an important factor affecting the growth of malignant pulmonary nodules.This study intends to provide a research direction for further searching for the essential causes of the growth of pulmonary.Part Four The effect of tumor-associated macrophage cytokines on the progression of lung carcinoma in situ to invasive carcinomaObjective:The tumor tissue is composed of variable numbers of cancer cells and stromal cells,and tumor-associated macrophages（TAM）,which together create a specific tumor microenvironment critical for tumor progres-sion.This study aimed to investigated the changes of TAM including the expression levels of cytokines from adenocarcinoma in situ（AIS）to invasive adenocarcinoma（IAC）of the lung.Methods:A total of 32 consecutive resected cases of pulmonary nodule were enrolled.Their pathological characteristics were collected after the thoracic surgical progression and divided into three groups including AIS,minimal invasive adenocarcinoma（MIA）and IAC.The expression level of the cytokines IL-6,IL-10,IL-12a,IL-12b,M-CSF,monocyte chemoattractant protein-1（MCP-1 also known as CCL2）,IFN-gramma-,were analyzed by Real Time PCR.Results:1.In this study,4 cases of benign nodules and 28 cases of malignant pulmonary nodules were confirmed by postoperative pathology.Analysis of cytokine expression in cancer tissue and paracancer tissue in malignant group showed that the expression levels of IL-6,IL-10,IL-12a,IL-12b,M-CSF and MCP-1 in cancer tissue were significantly higher than those in paracancer tissue（P<0.001）.The expression levels of IL-6,IL-10,IL-12a,IL-12b,M-CSF,MCP-1 increased from AIS to IAC;In IAC,IL-6,IL-10,IL-12a,IL-12b,M-CSF,MCP-1were significantly higher compared with in MIA and AIS.However,the IFN-gramma was not significantly different.2.In non-invasive group M1liked cytokines were almost positively correlated with levels of M2liked cytokines.But in the invasive component,these cytokines were not more positively correlated with each other than in non-invasive group.The IFN-gramma had no significant correlation with other cytokines.In the process of pathological progression,MCP-1 in M1-like cytokines increased significantly in the minimal invasive group,and M-CSF increased significantly in the invasive adenocarcinoma group.These results indicated that although various cytokines were correlated with each other and gradually increased in the process of invasion,the expression of M2-related factors was more prominent,which may promote tumor progression3.Subgroup analysis was performed in invasive adenocarcinoma,and pulmonary nodule was divided into growth group and stable group,and the difference of cytokine expression between the two groups was analyzed.The results showed that the expression levels of IL-12b（P=0.032）and M-CSF（P=0.033）were significantly different between the two groups,while the expression levels of other cytokines were not significantly different（P<0.05）.Therefore,IL-12b and M-CSF may be closely related to the growth of early malignant pulmonary GGNs.4.In pre-invasive lung adenocarcinoma（AIS and MIA）,ROC curve analysis was used to compare cytokine expression between the two groups,and it was found that the area under the expression curve（AUC）of M-CSF was the largest,which was also found that there was significant difference in the expression level of M-CSF compared with benign lesions（P=0.004）.These results suggest that M-CSF may play an important role in the carcino-genesis and pre-invasive stage of the ground glass nodules.Conclusions:1.With the progressive progression of early lung cancer,M1-like cytokines and M2-like cytokines in tumor microenvironment are gradually increased and correlated with each other,but the role of M2-related cytokines may be more prominent.2.The abnormal increase of M-CSF expression level may be an early event of malignant transformation or pre-invasion of GGN.This study may provide a theoretical basis for further immunotherapy of early malignant ground glass nodules.