| BackgroundCervical cancer is the third largest malignant tumor in female cancers in the world after breast and colorectal cancer.According to reports,there were 604,127 new cases of cervical cancer worldwide in 2020,accounting for 6.6%of the number of female cancers.The number of patients who died from cervical cancer has reached 314,831,accounting for 7.7%of all female cancer deaths,and 85%of the cases occurred in developing countries.The number of new cervical cancer patients in my country is about 135,000 each year,accounting for 28.8%of the global incidence.According to estimates by the International Agency for Research on Cancer（IARC）,by 2030,approximately 710,000 new cervical cancer patients will be added each year,and approximately 380,000 women will die from cervical cancer.However,the cause of cervical cancer is clear,and it can be cured through early screening,early detection of precancerous lesions,and timely treatment.Cervical squamous intraepithelial lesion（SIL）or cervical intraepithelial neoplasia（CIN）is a group of precancerous lesions closely related to invasive carcinoma of the cervix.Active treatment of cervical intraepithelial lesion to prevent cervical Cancer has been widely recognized.Cervical conization has become the most important treatment method due to its simple operation,less trauma,and good therapeutic effect.The prognosis of patients treated by cervical conization has also become closely watched by both doctors and patients.The problem.Although cervical conization has achieved a high success rate,it can cause complications such as bleeding,infection,cervical stenosis,cervical insufficiency,premature rupture of membranes during pregnancy,low birth weight infants,premature delivery,and anxiety,etc.Psychological impact;at the same time,about 5%to 25%of patients will have persistent or recurring lesions after cervical conization.Therefore,it is necessary to find an exact method to predict the negative factors of conization pathology to avoid unnecessary treatment and possible complications.Although HPV vaccine and cervical cancer screening programs have been carried out in China,due to the large population and uneven distribution of health resources in my country,cervical cancer is still the most common gynecological malignant tumor in China.For early cervical cancer,surgery and postoperative adjuvant treatment are the main treatment methods;for locally advanced cervical cancer patients,concurrent radiotherapy and chemotherapy are the main methods;for advanced or metastatic patients,platinum-containing chemotherapy or combined anti-vascular targeted therapy It is the main treatment.These treatments have reduced the mortality of cervical cancer patients,but their effectiveness has now reached a plateau.However,there are still some patients who relapse and metastasize after initial treatment,and some patients are already at an advanced stage at the time of diagnosis.At present,for patients with recurrence,metastasis,or advanced cervical cancer,the effects of chemotherapy and radiotherapy are not satisfactory,and their prognosis is still very poor.The median overall survival is about 17 months,and the 5-year survival rate is less than 20%.Therefore,for patients with recurrence,metastasis or advanced cervical cancer,new treatment methods are urgently needed to improve the survival rate.Tumor immunotherapy has become a new type of treatment after surgery,chemotherapy,radiotherapy and targeted therapy.In recent years,the targeted inhibition of the PD-1 axis for the treatment of cancer has garnered a lot of attention.PD-1 is a co-inhibitory cell-surface receptor,expressed mainly in B-and T-cells,that acts to restrain T-cellmediated immune responses when activated by its ligands,PD-L1 or PD-L2.Early clinical trials showed promising results with durable anti-tumor immune responses,which led to the approval of multiple PD-1-and PD-L1-targeting monoclonal antibodies for therapeutic use in various cancer types,including cervical cancer.The PD-1 inhibitor pembrolizumab is approved for the second-line treatment of PD-L1-positive recurrent or metastatic cervical cancer.However,from the results of Keynote-028 and Keynote-158 clinical studies,the objective response rate of pembrolizumab in PD-L1-positive cervical cancer is less than 20%.This shows that the efficacy of blocking a single immune checkpoint is not ideal.Therefore,it is necessary to find the mechanism of anti-PD-1 immunotherapy resistance,for cervical cancer patients,especially PD-L1-negative patients,to find new therapeutic targets and strategies to improve the efficacy of immunotherapy.PD-L1,also known as B7-H1,belongs to the B7 family.Currently,the B7 family includes 10 members,such as PD-L1,PD-L2,B7-H3（B7 homolog 3）and B7-H4（B7 homolog 4）.B7-H3,also known as CD276,is a type I membrane protein with a high degree of homology with other members of the B7 family.B7-H3 is abnormally highly expressed in various types of tumor cells,tumor infiltrating lymphocytes and tumor vascular endothelial cells.B7-H3 can not only stimulates the activation of T cells,but also inhibits the function of T cells.Because B7-H3 is involved in tumor immunity,chemotherapy resistance,tumor metastasis,etc.,there are already ongoing clinical studies targeting B7-H3.B7-H3 is highly expressed in cervical cancer,can promote the proliferation and migration of cancer cells,increase invasion and metastasis,and is an independent predictor of poor prognosis.Although the expression of B7-H3 in cervical cancer has been reported in previous studies,its correlation with PD-L1 is unclear.In addition,the number of patients included in previous studies was relatively small,especially those with adenocarcinoma.Therefore,the relationship between B7-H3 and histological type and survival outcome is unclear.B7-H4,first discovered in 2003,is another type I membrane protein in the B7 superfamily and has immunosuppressive effects.Although the expression of B7-H4 is restricted in normal tissues,it is expressed at high levels in a variety of tumors and has high tumor specificity.A number of studies have shown that the up-regulation of B7H4 expression in cancer is closely related to poor prognosis.After blocking PD-1 and B7-H4 at the same time,a strong synergistic anti-cancer effect was observed in vitro,indicating that blocking the B7-H4 pathway may improve the therapeutic effect of existing anti-PD-1 antibodies.However,the expression of B7-H4 in cervical cancer,its relationship with PD-L1 and its prognosis remain unclear.The purpose of this project is to explore the factors related to the negative pathology in conization of cervix after biopsy,to explore the expression of immune checkpoints B7-H3 and B7-H4 in cervical cancer and precancerous lesions,and the relationship with the clinical pathological characteristics,PD-L1 and prognosis,providing a basis for new strategies for immunotherapy of cervical cancer.Part ⅠAnalysis of the related factors of negative pathology after conization of cervical squamous intraepithelial lesionsObjective:To analyze the clinical factors related to negative pathology of cervical squamous intraepithelial lesions diagnosed by biopsy after cervical conization,and to explore the correlation between immune checkpoint B7-H4 and T cell subsets and negative conization.Methods:The clinical data of 807 patients who underwent cervical conization for cervical intraepithelial lesions in Qilu Hospital of Shandong University from July 2005 to December 2013 were retrospectively analyzed.The data were collected from the colposcopy computer database and the hospital patient database.We collected data including patient’s age,menopause,depth of conization,histological grade of biopsy,histological grading and margin status of conization specimen,gland involvement,etc.;patients with conization pathological diagnosis no lesion are regarded as pathologically negative in conization.Using the propensity scoring system,75 patients（experimental group-conization pathology negative）were selected to match 75 patients with SIL（pathologically positive in conization）confirmed by conization pathology（control group）.The nine matched factors were the patient’s age,menopause,liquid-based cytology（TCT）,biopsy pathological diagnosis,HPV status,resection margin status,gland involvement,colposcopy impression,and cervical conization method.Immunohistochemistry and immunofluorescence staining methods were used to detect B7-H4,regulatory T cells（CD4+Foxp3+）,CD4+T cells,CD8+ T cell and interferon-γ;20 patients with chronic cervicitis were selected as controls.Chi-square test or Fisher exact probability method were used for the analysis of categorical data.Multivariate logistic regression model was used to determine independent prognostic factors.Results:We collected 807 patients who underwent cervical conization after cervical biopsy diagnosis;the age range was 21-62 years,and the average age was 38.8±8.0 years.Among 807 conization specimens,104 cases（12.9%）had no intraepithelial lesions.Menopause,Other High-risk（exclude 16,18）HPV infection,unsatisfactory colposcopy,no gland involvement in the biopsy specimen,and conization depth ≤18mm are significantly related to absent intraepithelial lesions in the conization tissue.This data suggests that patients with CIN2 confirmed by cervical biopsy,TCT with no intraepithelial lesions and cancer cells（NILM）,atypical squamous cells（ASC）or low-grade squamous intraepithelial lesions（LSIL/CIN1）,and patients receiving LEEP treatment are less likely to have lesions after conization.Logistic multivariate regression analysis showed that CIN2 in cervical biopsy were independent predictors of absent lesions in conization.The positive expression rates of B7-H4 in CIN2 and CIN3 were 11.1%and 46.7%,respectively,but there was no expression in CIN1 and chronic cervicitis.The positive expression rate of B7-H4 in the biopsy specimens of the conization pathology-negative group was 17.3%（13/75）,and the positive rate in the biopsy specimens of the conization pathology positive group was 38.7%（29/75）,the difference was statistically significant（P<0.001）.The number of CD8+T cells,INF-γ and INF-γ+CD8+T cells in the B7-H4-negative group was significantly higher than that in the B7-H4-positive group.However,the number of Treg cells（CD4+Foxp3+）in the B7-H4-positive group was significantly higher than that in the B7-H4-negative group.There was no significant difference in the number of CD4+T cells between the two groups.These data indicate that B7-H4 is related to inhibitory signals in the tumor microenvironment of the cervix.Conclusion:About one-tenth of the biopsy-confirmed HISL patients have no lesions after cervical conization.Among them,about 20%of patients with CIN2 confirmed by biopsy have no lesions after cervical conization,which indicates that patients with CIN2 can choose close follow-up observation and reassessment.B7-H4 in HSIL inhibits T cell function and local anti-tumor immune response in the cervix.Part ⅡThe expression and clinical significance of B7-H3 and B7-H4 in cervical cancer Objective:To explore the expression patterns of B7-H3 and B7-H4 in cervical cancer,and to determine their correlation with PD-L1,clinicopathological characteristics and prognosis of patients with cervical cancer.Methods:Patients with cervical squamous cell carcinoma（N=406）treated in Qilu Hospital of Shandong University from January 2012 to December 2015 and endocervical adenocarcinoma from January 2008 to December 2017（N=146）were included in this study.The following clinicopathological data were recorded:age at diagnosis,tumor histological type,degree of differentiation,depth of tumor invasion,paramatrial involvement,lymphatic vessel space invasion（LVSI）,lymph node metastasis,postoperative adjuvant treatment（chemotherapy,radiochemotherapy or radiotherapy）,recurrence and death.Tissue microarray were made using paraffin specimens of 552 cases of cervical cancer after surgery,and the expression of B7-H4,B7-H3 and PD-L1 in the tissues was detected by immunohistochemical sstainings.The chi-square test was used to determine the association between categorical variables,the Kaplan-Meier method was used to draw the survival curve and the log-rank test was used for comparison.Results:The positive rate of B7-H3 expression in tumor cells was 32.1%,and the positive rate in stromal cells was 92.9%.In stromal cells,B7-H3 was mainly expressed in fibroblasts,macrophages and vascular endothelial cells.Co-expression of B7-H3 with PD-L1 was observed in 21%of the samples,while one or the other was expressed in 41.7%of the samples.The positive expression rate of B7-H3 in squamous cell carcinoma was 36.0%,which was significantly higher than the 21.2%in adenocarcinoma（P=0.001）.The positive rate of B7-H3 in patients with lymph node metastasis was 40.5%,which was significantly higher than 29.9%in patients without lymph node metastasis（P=0.032）.Compared with the PD-L1-negative group（22.8%）,the positive expression rate of B7H3 in the PD-L1-positive group was higher（40.7%）,indicating that the expression of B7-H3 and PD-L1 was positively correlated.The positive rate of PD-L1 in squamous cell carcinoma（58.4%）was higher than that in adenocarcinoma（32.9%）（P<0.001）;the positive rate in poorly differentiated tumors（43.5%）is higher than that in wellmoderately differentiated tumor（55.7%）（P=0.007）.Different from the expression patterns of B7-H3 and PD-L1,B7-H4 expressed in tumor cells,and the expression of B7-H4 was not detected in mesenchymal cells.252 cases（45.6%）were B7-H4 positive.The positive rate of B7-H4 in squamous cell carcinoma（52.0%）was significantly higher than that in adenocarcinoma（28.1%）（P<0.001）.The expression of B7-H4 and B7-H3 was positively correlated.The positive rate of B7-H4 in B7-H3-positive specimens（54.2%）was significantly higher than that of B7-H3-negative counterparts（41.6%）（P=0.005）.The expression of B7-H4 was not significantly correlated with the expression of PD-L1.In the entire study cohort,the expression of B7-H4 was not associated with the prognosis of patients.In the PD-L1-negative subgroup,patients with B7-H4-positive tumors have more favorable prognosis than those with B7-H4negative tumors,with an HR of 0.36 and a 95%confidence interval（0.17-0.77）.In univariate survival analysis,patients with B7-H3 positive in tumor cells,FIGO IIA1 stage,paramatrial involvement,deep stromal infiltration（≥1/2）,and lymph node metastasis have shorter recurrence-free survival（RFS）and overall survival（OS）.In the multivariate analysis,B7-H3 positivity in tumor cells was an independent predictor of shorter RFS（HR 2.1,95%CI 1.2-3.6,P=0.006）and OS（HR 1.8,95%CI 1.1-3.0,P=0.023）.The distribution of B7-H3 in the cervical adenocarcinoma Silva system:the positive rate in pattern A is 0%,17.1%in pattern B,and 27.9%in pattern C.B7-H3 had the highest positive rate in pattern C,and there was a significant difference among three groups（P=0.020）.Compared with patients with B7-H3-negative tumor,patients B7H3-positive adenocarcinoma had shorter RFS（P=0.012）and OS（P=0.007）.Conclusion:B7-H3 expressed in both tumor cells and stromal cells of cervical cancer tissues,and B7-H4 expressed in tumor cells.The positive rates of B7-H3 and B7-H4 in squamous cell carcinoma are higher than those in adenocarcinoma.The expression of B7-H3 in tumor cells is positively correlated with the expression of PD-L1 and B7-H4,while B7-H4 was not associated with the expression of PD-L1.The positive rate of B7H3 in tumor cells is higher in PD-L1-positive and lymph node metastasis tumors;B7H3 expression is a poor independent predictor.B7-H4 was not associated with the prognosis of cervical cancer patients in the entire study cohort,but in the PD-L1negative subgroup,patients with B7-H4-positive tumor had a better prognosis.Therefore,B7-H4 may be a prognostic factor and potential therapeutic target for patients with PD-L1-negative cancer,and the combined blocking of B7-H3 and PD1/PD-L1 may be a new strategy for immunotherapy of cervical cancer.PART ⅢThe efficacy of combined blockage of B 7-H3 and PD-1 in animal models of cervical cancerObjectives:To observe whether the anti-tumor effects of the combined blocking of B7-H3 and PD-1 can play a synergistic effect,and whether it can improve the efficacy of anti-PD-1 immunotherapy in mouse models of cervical cancer.Methods:Cervical cancer xenograft models were established in mice with normal immune function,and combined blocking B7-H3 and PD-1 treatment,blocking B7-H3 treatment,blocking PD-1 treatment and isotype antibody control were given,respectively.The tumor growth and volumes in each group were measured and compared.Results:The tumor volumes of the blocking B7-H3 treatment group and the blocking PD-1 treatment group were smaller than that of the isotype control group（P<0.01）,and the tumor volume of the combined blocking B7-H3 and PD-1 group was smaller than that of blocking alone B7-H3（P<0.01）or the PD-1 group（P<0.01）,and alao was significantly smaller than the isotype control group（P<0.001）.Conclusions:The combined blockage of B7-H3 and PD-1 has a synergistic effect,and the anti-tumor effect is more significant than that of blocking one of the immune checkpoints alone.Combined blockage of B7-H3 and PD-1 may be a new strategy for immunotherapy of cervical cancer. |
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