| Part Ⅰ Comparative study of clinicopathological characteristics and prognosis between combined and pure small-cell lung cancer(SCLC)after surgical resectionBackground:Histologically,SCLC are classified as pure(P-SCLC)and combined subtypes(C-SCLC).Currently,due to the low detection rate of C-SCLC,the prognostic factors for C-SCLC are unclear.Furthermore,so far,there is still no consensus on the treatment of C-SCLC in clinical practice.This study aims to compare the difference of clinicopathological characteristics and prognosis between P-SCLC and C-SCLC,and analyze the effects of different treatment methods on prognosis of the two histological subtypes in SCLC.Methods:Between July 2005 and April 2016,the clinical medical records of 297 postoperative patients with pathologically confirmed SCLC were collected and the corresponding postoperative pathological sections of included patients were reviewed to confirm diagnosis.The clinicopathological characteristics,treatment history,postoperative disease-free survival(DFS),overall survival(OS),and prognostic factors of P-SCLC and C-SCLC patients were compared and analyzed.Results:A total of 297 SCLC patients were included in this study.After reviewing the postoperative pathological sections,46 cases(15.5%)were found to be C-SCLC,and 251 cases(85.5%)were P-SCLCs.The average age of C-SCLCs was a little higher than that of P-SCLCs(59.65±8.72 vs.56.56±10.12;P=0.053).More patients had a history of smoking in C-SCLC(78.3%vs.63.3%;P=0.074).No significant differences of DFS and OS were identified between P-SCLCs and C-SCLCs.The 5-year DFS and OS rate for P-SCLCs and C-SCLCs was 52.5%vs.47.6%(P=0.944)and 65.1%vs.56.7%(P=0.683),respectively.For P-SCLC,clinical stage,and an intervention of prophylactic cranial irradiation(PCI)were independent factors that affected OS.In case of C-SCLCs,performing sublobectomy was an independent risk factors for poor prognosis.Conclusion:No significant differences in clinical characteristics and outcome between C-SCLCs and P-SCLCs were identified.But the factors influencing the prognosis of the two subtypes were slightly inconsistent.For C-SCLCs,the extent of resection had a greater impact on survival,thus the lobectomy combined with systemic lymph node dissection should be performed as extensively as possible.In addition,PCI was beneficial to improve the OS rate of SCLC.Part Ⅱ The influence of immune cell composition and abundance in the tumor microenvironment on the overall survival(OS)of patients with SCLCBackground:Small cell lung cancer(SCLC)is a refractory malignancy,and there has been no significant improvement in the treatment of SCLC for more than 30 years.Although immunotherapy improves overall survival(OS)in patients with SCLC,only a small percentage of patients could benefit.Currently,the understanding of the tumor immune microenvironment(TIME)of SCLC remains inadequate and has become a major obstacle to expanding the population to benefit from immunotherapy.Previous studies have shown that the immune microenvironment plays an important role in the occurrence and development of tumors,the response to immunotherapy and the prognosis of patients.However,different immune cells could have different effects on tumors,and the difference in the distribution proportion of immune cells in TIME may be an important factor leading to different clinical outcomes of SCLC patients.This study aims to analyze the influence of the distribution of different immune cell types in the TIME on the OS of postoperative SCLC patients,and to establish a prognostic prediction model based on the immune cell types that significantly affect the prognosis of SCLC patients,and to explore new therapeutic direction for immunotherapy.Method:SCLC transcriptome sequencing data were extracted from public data in the published literature.Based on the characteristic gene expression profiles of individual immune cells isolated in vitro,the transcriptome data were deconvolved using the CIBERSORT machine algorithm to obtain the relative proportion and absolute quantitative abundance of 22 types of immune cells defined and differentiated by cytologic types and functional status.Univariate Cox regression analysis was preliminarily performed to determine the relationship between the distribution of different types of immune cells and OS.Seventy-seven SCLC patients with follow-up information were divided into high infiltration group and low infiltration group according to the best cut-off value determined by X-tile software for each type of immune cells.Kaplan-Meier method with log-rank test was used to compare the OS.The immune cell types in univariate analysis whose abundance had a significant impact on OS were included in the multivariate Cox regression model.Meanwhile,LASSO regression was also used to screen immune cell types to construct the Cox regression model.Then we established risk score based on the best model.The Kaplan-Meier curves were drawn to measure the ability of the risk score to discriminate patients with different prognosis.Results:Univariate Cox regression analysis showed that high relative proportion of follicular helper T cells(Tfh)is protective for OS(HR 1.30e-05,95%CI:3.2e-09-0.52,P=0.008),while high absolute abundance of resting NK cells(HR:6.4E+08,95%CI 17-2.4E+16,P=0.023)and high relative proportion of γΔT cells(HR 4E+03,95%CI 6.5-2.5E+06,P=0.011)and resting NK cells(HR 1.2E+10,95%CI 200-7.5E+17,P=0.011)were hazardous for OS.Log-rank test between groups divided by the optimal cutoff value of CIBERSORT absolute quantitative results also showed that high abundance of resting NK cells and γΔT cells is a risk factor for shorter OS(resting NK cells:P=0.029,γΔT cells:P=0.021),while high abundance of Tfh cells(P<0.001),CD8+T cells(P=0.002),naive B cells(P=0.011),M1 macrophages(P=0.017)and resting CD4+ memory T cells(P=0.031)were protective for OS.Comparsion of groups stratified by relative proportion showed that the high proportion of Tfh cells(P=0.001),and CD8+T cells(P=0.002)and naive B cells(P=0.02)were favorable for OS,while high proportion of γΔT cells(P<0.001)and M2 macrophages(P=0.002)was risk factors for shorter OS.In multivariate analysis,both relative and absolute quantitative results indicated that high proportion of resting NK cells and γΔT cells were independent risk factors for OS.The Cox regression model constructed by variables screened by LASSO regression combined with backward stepwise was better than the Cox regression model including variables screened by univariate analysis.The area under the curve(AUC)of the optimal model for predicting one-year,three-year and five-year survival rates was 0.852,0.816 and 0.796,respectively.Median OS(mOS)was significantly shorter for patients assessed as high risk based on the optimal model than for those assessed as low risk(mOS:15 vs.48 months,HR 5.25,95%CI 2.68-10.30,P=9.53e-08).Conclusion:The abundance of resting NK cells and y△T cells was significantly negatively correlated with the OS of SCLC patients in all Cox proportional hazard models constructed with different variable screening methods,indicating high proportions of resting NK cells and y△T cells in the TIME were independent risk factors for poor prognosis in SCLC patients.The risk score based on the Cox regression model constructed with the absolute abundance or relative proportion of resting NK cells andγ Δ T cells can discriminate patients with significant different OS.Part Ⅲ Correlation between expression of immune cell-related molecular markers and prognosis in tumor immune microenvironment(TIME)of small cell lung cancer(SCLC)Background:The difference in prognosis of patients with small cell lung cancer(SCLC)mainly depends on the staging,but it has been found that patients with similar stage and treatment regimen could have distinct prognosis by clinical observation.Thus it is worth of studying the influence of related biological mechanisms on prognosis in SCLC patients.The results of the analysis based on the open database in the previous chapter suggest that the tumor immune microenvironment(TIME)is a vital factor affecting the outcome of patients with SCLC This part of the study aims to further analyze the expression of specific immune cell-related molecular markers in TIME and their correlation with postoperative disease-free survival(DFS)and overall survival(OS)in SCLC patients,and further to reveal the immunomicroenvironmental biomarkers with potential therapeutic and prognostic significance.Methods:Forty-eight paired formalin-fixed paraffin-embedded(FFPE)samples of SCLC patients with distinct prognosis were retrieved from the pathological specimen repository.We detected the mRNA expression of molecular markers of immune cells such as B cells(CD19,CD20),T cells(CD4,CD8,FOXP3),NK cells(CD69,NKG7,NCR1)using Nanostring nCounter technology.Vital cytokine and their receptors such as IL6,IL6R,IL17RB which had a close relationship with gamma delta(γΔ)T cells and resting NK cells whose abundance significantly influenced OS based on the public data analysis were also detected.We compared the expression of all detected molecules among groups with distinct prognosis,stages.Univariate survival analysis and multivariate Cox regression analysis were performed for each marker to identify and screen the immune cell types and related immune effector molecules that had significant influence on prognosis.Results:Median OS was 19.1 months(95%CI 15.4-40.5 months)in the group with poor prognosis,while it was not achieved in the group with good prognosis.The difference in OS between the two groups was significant(P=2.1e-09).However,there were no significant differences in clinical characteristics including gender,age,proportion of smoking patients,clinical stage,lymph node status,treatment and surgical methods between the two groups.However,when comparing the expression of immun-related molecular markers between the two groups,it was found that the expression of IL17RB was significantly higher in the poor prognosis group(P=0.015),while the expression of interferon γ(IFNG)and perforin(PRF1)were significantly lower in the poor prognosis group(IFNG:P=0.045;PRF1:P=0.013).Univariable Cox regression analysis confirmed that high expression of IL17RB was hazardous for shorter DFS,while high expression of CD8A,NCR1,NKG7,PRF1,GZMB,GZMH and IFNG were favorable for DFS.Multivariable Cox regression analysis which including clinicopathological and transcriptional data identified high expression of IL6 mRNA was hazardous for both DFS and OS independent of clinicopathological factors,while high expression of cytotoxic cell markers and immune effector molecules were protective for DFS and OS.Conclusion:Abundance of NK cells and CD8+T cells in TIME significantly affected the postoperative DFS of patients with SCLC,the content of NK cells also significantly affected the OS of SCLC patients.Patients with high expression of IL6 and IL17RB mRNA had significantly shorter postoperative DFS,and high expression of IL6 could significantly influence DFS and OS independently of other clinicopathological prognostic factors.Personalized or targeted immunotherapy based on the characteristics of the TIME of SCLC may improve the current treatment dilemma. |
