A Retrospective Study On Surgical Treatment Of Synchronous Multiple Pulmonary Nodules And A Preliminary Study On Their Immune Microenvironment

Objective: This study reviewed and analyzed the surgical diagnosis and treatment strategies of synchronous multiple pulmonary nodules,and preliminarily explored the immune microenvironment of multiple primary lung adenocarcinoma with multiple pulmonary nodules.Methods: This study is a retrospective study,which consists of four parts.The first part retrospectively collected the clinical data of patients with multiple ground-glass opacities(GGO)who underwent simultaneous surgery in the department of thoracic surgery,Wuhan Tongji Hospital from November 2015 to May 2019;The second part is mainly for the patients with multiple GGO who cannot tolerate multiple lesion resection.The retrospective analysis is made on the clinical case data of such patients who received electromagnetic guided bronchoscopic microwave ablation combined with uniportal video-assisted thoracoscopic surgery(Uni-VATS)in the department of thoracic surgery,Wuhan Tongji Hospital from December 2019 to June 2021.The first two parts of the study focus on the clinical characteristics and perioperative conditions of the patients,the safety and effectiveness of the two surgical strategies were analyzed;The third part retrospectively collected the data of patients with different EGFR mutations who were pathologically diagnosed as synchronous multiple primary lung adenocarcinomas(SMPLA)and underwent surgical resection in our hospital from March 2015 to December 2019,and focused on the clinical features,surgical results,recurrence free of survival(RFS)and overall survival(OS)of the patients;In the fourth part,immunohistochemistry technique was performed to stain related immune markers(CD8,CD103,CD20,CD138,CD4,FOXP3,CD68,CD163,PD-1 and PD-L1)and TGF-β from 15 patients with pure GGOLUAD and 15 patients with SN-LUAD tissue sections(Paired cohort),and then,the related markers with significant differences were verified on 10 patients(Verified cohort)with both pure GGO-LUAD and SN-LUAD.Results: In the first part of the study,65 patients with multiple ground-glass pulmonary nodules were finally included.Among them,48 patients underwent unilateral surgery at the same time,and 17 patients underwent bilateral surgery at the same time.Except for two patients who were converted to thoracotomy due to total thoracic adhesion,the other patients underwent thoracoscopic surgery,and there were no serious complications and deaths during the perioperative period;In the second part of the study,a total of 29 patients with multiple GGO underwent ENB-guided microwave ablation combined with Uni-VATS.A total of 118 GGO lesions were included,except for 5 untreated lesions,of which 46 lesions were treated by microwave ablation and 67 lesions were removed by surgery.Only two patient developed postoperative pneumothorax and subcutaneous emphysema,without other serious complications and surgically related deaths.During the follow-up period,no metastasis or recurrence occurred in all patients;In the third part of the study,70 patients were finally included.There were 161 tumors,84.4% of which were GGO lesions.The mutation rate of mixed GGO(m GGO)is significantly higher than that of pure GGO(p GGO)and solid nodules(SN);the mutation rate of invasive adenocarcinoma is significantly higher than that of other histology subtypes;the mutation rate of lesions >20 mm was significantly higher than that of ≤20 mm.However,there is no significant difference in the mutation rate of specific driver gene between different radiological features,pathological characteristics and sizes.After a median follow-up time of 29 months,the 3-year OS and RFS were 94.4%and 86.0%,respectively.The fourth part of the study showed that compared with SN-LUAD,only the infiltration of TAMs and Treg cells was significantly lower in GGO-LUAD.The infiltration of the remaining immune cells including CD8+ T cells,CD4+ T cells,CD103+T cells,CD20+ B cells and CD138+ Plasma cells in GGO-LUAD,although relatively low,was not significantly different.Meanwhile,the expression of TGF-β was significantly higher in SN-LUAD.And the above results have also been confirmed in the verified cohort.Moreover,there was no significantly difference in PD-L1 expression in GGO-LUAD compared to SN-LUAD both in the paired cohort and verified cohort.Conclusion: A high rate of multiple GGOs in the lungs are malignant.And when lung function is sufficient for patients to underwent surgeries,the simultaneous unilateral or bilateral thoracoscopic resection is suggested,which will not increase the risk of postoperative complications.For patients who cannot tolerate multiple lesion resection,ENB-guided microwave ablation combined with Uni-VATS is safe and feasible in patients with multiple pulmonary nodules suspected of having multiple primary lung cancers;Synchronous multiple lung adenocarcinomas with predominantly multiple GGO should be considered as SMPLA,and surgery may be aggressively performed for these patients due to a good prognosis;In multiple primary lung adenocarcinoma with multiple lung nodules,GGO-LUAD showed lower immune activity compared with SN-LUAD.Part Ⅰ: Single-stage resection of multiple pulmonary ground-glass opacities: A clinical analysisObjective: To summarize our experience of surgical resection of multiple ground-glass opacity(GGO)in recent years.Methods: Clinical data of patients who underwent one-stage resections of multiple GGO from November 2015 to May 2019 in our hospital were collected,including 13 males and 52 females at an average age of 56.0±9.4 years.The clinical effects and pathological types of GGO were evaluated.The safety and feasibility of single-stage resection of multiple pulmonary nodules were analyzed.Results: Time interval from first discovery to surgery was 8-1447(236.5±362.4)days.There were 48 patients with unilateral surgery and 17 patients with bilateral surgery during the same period.Except for 2 patients who underwent open thoracotomy due to total thoracic adhesions,other patients underwent video-assisted thoracoscopic surgery(VATS).The mean postoperative hospital stay was 12.2±4.3 days.No severe perioperative complication or death occurred.A total of 156 GGO lesions were resected,80 lesions were pure GGO,including 58(72.5%)malignant lesions and 22(27.5%)benign lesions,with an average diameter of 7.7±3.3 mm and 5.5±2.6 mm,respectively.Another 76 lesions were mixed GGO,including 69(90.8%)malignant lesions and 7(9.2%)benign lesions,with an average diameter of 13.6±6.6 mm and 7.7±3.5 mm,respectively.Conclusion: A high rate of multiple GGOs in the lungs are malignant.And when lung function is sufficient for patients to underwent surgeries,the simultaneous unilateral or bilateral thoracoscopic resection is suggested,and the sublobar resection or lobectomy methods can be adopted flexibly according to the clinical features of the lesion and the rapid pathological results,which will not increase the risk of postoperative complications.Otherwise,surgical resection should be given priority for pure GGO lesions with a diameter > 7.7 mm and mixed GGO lesions.Part Ⅱ: Electromagnetic navigation bronchoscopy-guided microwave ablation combined with uniportal video-assisted thoracoscopic surgery for multiple pulmonary ground-glass opacitiesObjective: An increasing number of patients are being diagnosed with multiple pulmonary nodules,but a consensus on the treatment of these patients is still lacking.The aim of this study was to investigate the safety and feasibility of a novel technique,electromagnetic navigation bronchoscopy(ENB)-guided microwave ablation combined with uniportal video-assisted thoracoscopic surgery(Uni-VATS),in patients with multiple ground-glass opacity nodules.Methods: The clinical,radiographic,surgical,and pathological data of patients with multiple pulmonary nodules who underwent ENB-guided microwave ablation combined with Uni-VATS from December 2019 to June 2021 were reviewed.Results: Eleven patients with multiple GGOs underwent ENB-guided microwave ablation combined with Uni-VATS,including 7 males and 22 females with a mean age of 52.8 ± 8.5(32-73)years.A total of 118 GGO lesions,except for 5 lesions only undergoing biopsy,46 of which were treated with microwave ablation and 67 of which were removed surgically.The average time for ablation of each lesion was 3.3 ± 0.7 minutes,with a median ablation power of 50W(10W-70W)and a median postoperative hospital stay of 8 days.Only two patient developed postoperative pneumothorax and subcutaneous emphysema and was successfully discharged from the hospital after symptomatic treatment.The success rate and efficiency of microwave ablation under ENB guidance were 100%,with no other serious complications or procedure-related deaths occurring.No metastasis or recurrence occurred in any patients during the follow-up period.Conclusions: ENB-guided microwave ablation combined with Uni-VATS is safe and feasible in patients with multiple pulmonary nodules suspected of having multiple primary lung cancers.By treating multiple lesions at one time,not only can the lung lesions be effectively treated,but also the healthy lung tissue of the patient can be preserved to the maximum extent.Part Ⅲ: Clinical features and surgical treatment of synchronous multiple primary lung adenocarcinomas with different EGFR mutationsObjective: With the popularity of lung cancer screening and advances in imaging technology,more and more synchronous multiple primary lung adenocarcinomas(SMPLA)are being diagnosed clinically,however,the clinical characteristics and prognosis of SMPLA with different EGFR mutations remains unclear.We aimed to explore clinical features and surgical outcomes of these patients to aid in the diagnosis and treatment of SMPLA.Methods:Medical records of patients with different EGFR mutations who have been diagnosed as SMPLA and underwent surgical resection from March 2015 to December 2019 were retrospectively analyzed.Clinical characteristics,surgical outcomes,recurrence-free survival(RFS)and overall survival(OS)were investigated.Results:A total of 70 patients(68.6% female and 77.1% non-somkers)were included.Total of 161 lesions in all patients,84.4% were ground-glass opacity(GGO)lesions.EGFR mutations were detected in 108 lesions,most of which were L858R(35.4%)and 19Del(20.5%).The mutation rate of mixed GGO(m GGO)is significantly higher than that of pure GGO(p GGO)and solid nodules(SN);the mutation rate of invasive adenocarcinoma is significantly higher than that of other histology subtypes;the mutation rate of lesions >20 mm was significantly higher than that of ≤20 mm.However,there is no significant difference in the mutation rate of specific driver gene between different radiological features,pathological characteristics and sizes.After a median follow-up time of 29 months,the 3-year OS and RFS were 94.4% and 86.0%,respectively.Conclusions:A high discordance of EGFR mutations was identified between tumors in patients with SMPL,so EGFR can be used as a routine detection method for postoperative diagnosis of SMPLA.Synchronous multiple lung adenocarcinomas with predominantly multiple GGO should be considered as SMPLA,and surgery may be aggressively performed for these patients due to a good prognosis.Part Ⅳ: Distinct cellular immune profiles in lung adenocarcinoma manifesting as pure ground glass opacity versus solid nodulesObjective: At present,a large number of research results show that ground glass opacity featured lung adenocarcinomas(GGO-LUAD)display more indolent biological behavior than solid nodule featured lung adenocarcinomas(SN-LUAD)and have an excellent prognosis,but the cellular immune characteristics of GGO-LUAD remain poorly understood.The purpose of this study was to analyze the difference between the infiltration of immune cells and the expression of related immune molecules in GGO-LUAD and SNLUAD.Methods: Immunohistochemistry technique was performed to stain related immune markers(CD8,CD103,CD20,CD138,CD4,FOXP3,CD68,CD163,PD-1 and PD-L1)and TGF-β from 15 patients with pure GGO-LUAD and 15 patients with SN-LUAD tissue sections(Paired cohort),and then,the related markers with significant differences were verified on 10 patients(Verified cohort)with both pure GGO-LUAD and SN-LUAD.For localization analysis of CD68+ tumor-associated macrophages(TAMs)and FOXP3+ Terg cells in tumor areas,pure GGO-LUAD and SN-LUAD were also stained for simultaneous detection of pan-CK,CD68 and FOXP3 by multiplex immunofluorescence.Results: In the paired cohort,compared with SN-LUAD,only the infiltration of TAMs and Treg cells was significantly lower in GGO-LUAD.The infiltration of the remaining immune cells including CD8+ T cells,CD4+ T cells,CD103+ T cells,CD20+ B cells and CD138+ Plasma cells in GGO-LUAD,although relatively low,was not significantly different.Meanwhile,the expression of TGF-β was significantly higher in SN-LUAD.And the above results have also been confirmed in the verified cohort.Moreover,there was no significantly difference in PD-L1 expression in GGO-LUAD compared to SN-LUAD both in the paired cohort and verified cohort.Conclusions: GGO-LUAD demonstrates an overall less active immune landscape as compared with SN-LUAD.TAMs and TGF-β may play an important role in the progression of GGO-LUAD.More importantly,PD-L1 expression in GGO-LUAD is comparable to that in SN-LUAD,indicating that there may be other reasons for the insensitivity of GGOLUAD to immunotherapy.