The Clinicopathological Significance Of PD-L1 Expression And Construction Of The B Cell Marker Gene Signature In Head And Neck Squamous Cell Carcinoma

Part one:The clinicopathological significance of PD-L1 expression in head and neck squamous cell carcinomaBackground:Programmed cell death ligand 1(PD-L1)expressed in head and neck squamous cell carcinoma(HNSCC)was used as a predictive biomarker for immunotherapy,and FDA recommended the combined positive score(CPS)for PD-L1 scoring in patients with HNSCC.Objective:There are few studies on the expression of PD-L1 in HNSCS using CPS as the standard.Meanwhile,the results of these existing studies are contradictory,and the positive expression rate of PD-L1 differs from related clinical factors.Therefore,it is still valuable to accurately analyze the expression of PD-L1 in HNSCS by the CPS assessment method.Methods:We retrospectively collected 119 patients with primary HNSCC diagnosed and operated in the Chinese Academy of Medical Sciences Cancer Hospital.Including 34 cases of oral cavity squamous cell carcinomas(OSCC),9 cases of oropharyngeal squamous cell carcinomas(oropharyngeal squamous cell carcinomas,OPSC),36 hypopharyngeal squamous cell carcinomas(HPSC),and 40 cases of laryngeal squamous cell carcinomas(LSC).Patients who received chemotherapy or radiation before surgery were included.Monoclonal antibody 22C3(Dako)was used for immunohistochemical staining and CPS score to evaluate the expression of PD-L1.And we analyzed the relationship between PD-L1 expression and clinicopathological features by Chi-square test or Fisher’s exact test.Results:1)When the CPS threshold was set to 1,the 107 HNSCC patients(89.9%)were positive for PD-L1 expression.PD-L1 expression was positive in 52 HNSCC patients(43.7%)when the CPS cutoff was set to 20.2)Tumor stage(P=0.022)and tumor site(P=0.004)were correlated with PD-L1 expression(CPS≥1),and non-diabetic patients(P=0.046)were correlated with positive PD-L1 expression(CPS≥20).Whether the cutoff value was 1 or 20,there was no significant correlation between PD-L1 expression and age(P=0.083,P=0.085).3)For 40 LSC samples,when the CPS threshold was set at 20,N stage was significantly correlated with the positive expression of PD-L1(P=0.042).4)When assessing PD-L1 expression in 36 HPSC samples,positive expression of PD-L1(CPS≥1)was associated with young adults(P=0.030)and patients without type 2 diabetes(P=0.040).When the CPS threshold was set to 20,the T1 stage(P=0.024)was associated with positive expression of PD-L1.5)Univariate Cox regression analysis showed no significant correlation between clinical features and OS or PFS.Clinical prognostic factors were assessed independently by a Log-rank test.However,no matter the threshold of CPS was set to 1 or 20,PD-L1 expression was not correlated with OS or PFS.Conclusions:We used 22C3 retrospectively to detect the expression of PD-L1 in HNSCC patients,and studied the relationship between PD-L1 expression and clinicopathological parameters in patients more likely to benefit from anti-PD-1/PD-L1 therapy.In our study,HNSCC with type Ⅱ diabetes were negatively correlated with the expression of PD-L1,which may be related to the immune mechanism involved in typeⅡ diabetes.The effect of immunotherapy in patients with type Ⅱ diabetes deserves further discussion.Part two:Comprehensive analysis of single-cell and bulk RNA-sequencing data identifies and verifies B cell marker genes signature that predicts prognosis in head and neck squamous cell carcinomaBackground:Although the classic TNM staging of head and neck squamous cell carcinoma(HNSCC)has been widely used in clinical diagnosis and to assist clinical treatment decisions,the definition of TNM staging of HNSCC is based solely on the tumor itself as a highly heterogeneous malignancy.It is not enough to accurately predict the prognosis of HNSCC patients.More and more studies have shown that various immune cells in the tumor microenvironment,especially B cells,play a crucial role in tumor development and prognosis.Objective:In this study,we aimed to develop a B-cell marker gene-related model to indicate the prognosis of HNSCC patients.Methods:We obtained 18 primary HNSCC samples of GSE164690 from the GEO database single-cell RNA-sequencing(RNA-Seq)and analyzed B cell marker genes of HNSCS.Transcriptome expression profile data of 499 TCGA-HNSCC primary sites were downloaded from UCSC-XENA as a test group.Four transcriptome expression data sets with clinical annotation and follow-up information were obtained from GEO and ArrayExpress databases as validation sets to verify the efficacy of the prognostic model.The expression profiles of GSE41613,GSE42743,GSE65858 and E-MTAB-8588 contained 97,74,270 and 108 HNSCC samples,respectively.We used univariate Cox regression,LASSO regression,and multivariate Cox regression to establish a prognostic risk model.According to the risk index score,patients were divided into high-risk and low-risk groups.Based on the independent factors obtained by multivariate Cox regression analysis,the 2-year,3-year and 5-year OS charts of TCGA-HNSCC patients were constructed.To further explain the biological functions of the target Genes,GO(gene ontology)analysis were conducted and the KEGG(Kyoto Encyclopedia of Genes and Genomes)approach was analyzed jointly.In addition,we analyzed stromal and immune cell scores in TCGA-HNSCC using the ESTIMATE algorithm,estimated the abundance of immune cell infiltration in high-and low-risk groups using the CIBERSORT algorithm,and analyzed the relationship between risk scores and tumor microenvironment.Expression differences between TCGA-HNSCC tumor tissues and matched normal tissues were analyzed using GEPIA2 from the TCGA normal and GTEx datasets.We collected tissue from 114 primary HNSCC diagnosed in the Cancer Hospital of Chinese Academy of Medical Sciences.Tissue microarrays(TMAs)were constructed from formalin-fixed paraffin-embedded(FFPE)samples.Immunohistochemistry(IHC)was performed by the automated system of Ventana BenchMark platform.Genes in the model and proteins encoded by immune checkpoints were stained,including FTH1,PD-1,CTLA-4,TIM-3,LAG-3,TIGIT,PD-L2 and PD-L1.Results:1)We comprehensively analyzed the Single cell RNA-Seqing data of HNSCC in GEO database and screened 145 B cell marker genes(BCMG).2)We identified seven genes(FCRLA,FTH1,LAT,S100A4,TMP1,TNFRSF18,and TXN)to establish B cell marker gene signature(BCMGS).3)BCMGS were established using the TCGA-HNSCC dataset and verified in four independent datasets.4)Multivariate Cox regression analysis identified this model as an independent prognostic factor,and we used this model to construct a prognostic nomogram with other clinical factors.5)Studies of immune profiles have shown that patients in the low-risk group exhibit immune cell infiltration.In addition,the low-risk group was characterized by a higher diversity of TCR and BCR,suggesting that low-risk patients may be more sensitive to immunotherapy.6)We assessed the expression of seven genes in BCMGS between TCGA-HNSCC tumor tissues and matched normal tissues using GEPIA2.The protein expression of FTH1 and TNFRSF18 was significantly highly expressed in tumor tissues compared with normal tissues.No significant correlation was found in the protein expression of FCRLA,LAT,S100A4,TMP1,and TXN between tumor tissues and normal tissues.7)Immunohistochemical examination showed that high expression of FTH1 was significantly correlated with OS difference(P=0.025).The expressions of TIM-3,LAG3,and PD-1 were associated with better OS in HNSCC patients.However,there was no statistically significant difference between PD-L1,PD-L2,CTLA-4,TIGIT,and prognosis.Conclusion:BCMGS is a promising prognostic biomarker in HNSCC that may help explain immunotherapy responses and provide a new perspective for future studies of HNSCC.