A Comparative Study Of Immune Microenvironment Differences In Small Cell Lung Cancer Based On Molecular Subtype Stratified By Lineage Transcription Factors

Part Ⅰ The Impact of Immune Microenvironment Differences on the Immunotherapy Efficacy of Small Cell Lung Cancer Based on Molecular Subtypes of Lineage Transcription FactorsObjective:Small Cell Lung Cancer(SCLC)is a refractory and highly aggressive form of lung cancer.After first-line chemotherapy,most patients relapse quickly and have a poor prognosis.Molecular subtypes by lineage transcription factors(TFs)have shown some heterogeneity and different therapeutic weaknesses,but the mechanisms behind are not yet clear.This study aims to explore the heterogeneity of Tumor Immune Microenvironment(TIME)under the molecular subtypes based on lineage transcription factors,as well as its implications for stratified immune therapy.Methods:(1)Transcriptome sequencing data of 81 SCLC patients published by George et al.were selected.The maximum Z-score value of mRNA expression levels of four key TFs,ASCL1,NEUROD1,POU2F3 and YAP1,was used as the molecular subtype of the sample.The following methods were used to quantify the TIME:a.Quantitative analysis of Tumor Infiltration Lymphocytes(TILs),including CIBERSORTx machine algorithm,T Cell Infiltration Score(TIS),gene CD8;b.Quantitative analysis of immune effector factors,including T-cell-inflamed Gene Expression Profiles(T-cell-inflamed GEP)scores,Cytotoxic Activity(CYT)score and Immune Checkpoints Signature(ICS)score.Kruskal-Wallis test was used to compare the expression differences of biomarkers of different molecular subtypes.(2)Using RNA-seq data of 17 patients before combination therapy in Roper et al.Phase II study(NCT02484404),Kaplan-Meier curve and Fisher exact test were used to compare the difference in prognosis and efficacy of combination therapy among different subtypes.Results:(1)The public data from George et al.included 81 SCLC samples,with the proportion being SCLC-A(n=33,40.7%),SCLC-N(n=16,19.8%),SCLC-P(n=10,12.3%),and SCLC-Y(n=22,27.2%).Three TILs quantitative analysis methods revealed CD8 T cells were enriched in SCLC-P subtype,higher than SCLC-A/N/Y subtypes,as seen in CIBERSORTx(p=0.032),TIS(p=0.032),and gene CD8(p=0.032).Additionally,CIBERSORTx showed high expression of monocytes in SCLC-Y subtype(p=0.028).Quantitative markers of immune effector T-cell-inflamed GEP score,CYT score,and ICS score were not significantly different between subtypes,but the above scores tended to be higher in SCLC-P subtype.(2)Roper et al.public data on SCLC sample types included 17 cases before combined immunotherapy,with the proportion being SCLC-A(n=10,58.8%),SCLC-N(n=5,29.4%),SCLC-P(n=1,5.9%),and SCLC-Y(n=1,5.9%).Patients with SCLC-P subtype had the highest proportion of Clinical Benefit(CB),and their therapeutic response was better than other subtypes(p=0.181).Kaplan-Meier survival curve demonstrated that SCLC-Y had the worst prognosis after combined immunotherapy(p=0.029).Conclusion:Based on lineage TFs,molecular subtype indicate heterogeneity in TIME and different responses to immunotherapy.Compared with the SCLC-A/N subtypes,the SCLC-P/Y subtypes showed enrichment of specific immune cells and a higher score of immune effector gene set,suggesting a more "hot" TIME.The specific molecular mechanism is worthy of further investigation.Part Ⅱ The Study on the Mechanism of Immune Microenvironment Differences in Small Cell Lung Cancer Based on Molecular Subtype of Lineage Transcription FactorsObjective:The purpose of this study was to validate the characteristics of TIME between different molecular subtypes of SCLC at the protein level,obtained from the above transcriptome public data.Methods:This study included 48 limited-stage SCLC surgical samples(July 2009April 2016)from Cancer Hospital of Chinese Academy of Medical Sciences.Nanostring nCounter technology was employed to measure mRNA expression of key TFs(ASCL1,NEUROD1,POU2F3,YAP1)based on Tissue Microarrays,and the Z-score was calculated to confirm the molecular subtype group.Observe the following molecular subtype heterogeneous characteristics:(1)Morphological characteristics:based on pathological morphology,it is divided into Classic and Variant group.(2)TIME characteristics:based on the analysis of transcriptome public data and combined with clinical practical application,CD8 and PD-L1 indicators were finally selected for immunohistochemical(IHC)verification.a.CD8 interpretation and analysis:10 regions were selected for each HE section(5 tumor centers and 5 tumor border areas),and the CD8 positive cell density was interpreted by image recognition software Qupath.Results were exported and compared the differences in CD8 positive cell density among different molecular subtypes.b.According to the CD8 T cell immune phenotype,it is divided into"immune desert"(low/no expression of CD8 positive cells in the tumor and fibrous interstitium adjacent to tumor),"immune exclusion"(CD8 positive cells do not directly contact tumor cells and express in the adjacent stroma),and "immune inflammation"(CD8 positive cells directly contact tumor cells).The immune phenotype of each case was interpreted.c.PD-L1 interpretation and analysis:the Tumor Proportion Score(TPS)was calculated manually by two professional pathologists,which is the percentage of positive tumor cells in the total number of tumor cells.A cut-off value of 5%was used as the positive expression standard to compare the PD-L1 positive expression rate among different molecular subtypes.The inter-group differences were compared by KruskalWallis test and Fisher exact test.Results:Molecular subtypes and proportion of 48 limited-stage SCLC surgical samples were as follows:SCLC-A(n=22,46%),SCLC-N(n=11,23%),SCLC-P(n=4,8%)and SCLC-Y(n=11,23%).(1)The SCLC-A and SCLC-N subtypes are mainly exhibiting a Classic morphology(with relatively small cells,unclear boundaries,and "salt and pepper" nuclear appearance),while the SCLC-P and SCLC-Y subtypes tend to exhibit Variant morphology(nest-like cell arrangement,distinct boundaries,larger cell nucleus,and some observable single small nuclei).(2)The overall CD8 positive cell density and PD-L1 positive rate of SCLC-A/N subtypes were lower than those of SCLC-P/Y subtypes(median overall CD8-positive cell density:SCLC-A/N/P/Y=1097.5,691.7,2905.6,2896.6,respectively,p<0.01;PD-L1 positivity rate:SCLC-A/N=0,SCLC-P=50%(2/4),SCLC-Y=36.4%(4/11),p<0.01),mainly showing an "immune desert" phenotype(SCLC-A:17/22,77.2%;SCLC-N:9/11,81.8%).The SCLC-P subtype comprised 50%"immune inflammation" and 50%"immune rejection" cases,with high CD8-positive cell density in the tumor center(median CD8-positive cell density in the tumor center:SCLC-A/N/P/Y=1498.4,583.3,4114.0,3991.4,respectively,p<0.01).The SCLC-Y subtype has the highest proportion(6/11,54.5%)of "immune exclusion" cases,with high CD8-positive cell density in the tumor margin(median CD8-positive cell density in the tumor margin:SCLC-A/N/P/Y=724.7,833.3,778.3,2270.1,respectively,p=0.005).Conclusions:Based on observations and comparisons of morphoIogy,CD8 T cell expression patterns,and PD-L1 positivity among molecular subtypes,we found that molecular subtypes have TIME characteristics that are similar to those observed at the transcriptome sequencing level.Specifically,SCLC-A and SCLC-N subtypes are tended to a Classic morphology and an "immune desert" phenotype,with lower CD8 T cell and PD-L1 positivity rates.SCLC-P and SCLC-Y subtypes mainly exhibit a Variant morphology,an "immune inflammation" and "immune exclusion" phenotype,and higher CD8 T cell and PD-L1 positivity rates.Based on these characteristics,a multidimensional pathological classification system based on the interaction between tumor substance and stroma can be established,which is helpful for developing stratified treatment strategies in clinical research and practice.