Effect Of (Chemo) Radiotherapy On Circulating Immune Cells In Cervical Cancer And Its Clinical Significance

Background:Cervical cancer is one of the most common malignant reproductive system tumors in women.For locally advanced cervical cancer,pelvic external beam radiation radiotherapy（EBRT）+concurrent chemotherapy+brachytherapy is the preferred treatment.However,the 5-year survival after chemoradiotherapy is only about 70%.Further research is needed to improve the prognosis of locally advanced cervical cancer.Immune system plays an important role in radiotherapy（RT）.Preclinical studies have demonstrated that RT alone controlled the tumors in immunogenic mice,but not in immune-deficient mice.At present,the tumor immunology field has mainly focused on local immune responses in the tumor microenvironment（TME）.However,beyond impact on TME,cancer per se and anti-tumor treatment can induce dynamic changes of the systemic immune components and functions.RT can promote or inhibit the systemic immunity.Understanding systemic immune consequences induced by RT is important for designing strategies that augment rather than impede anti-tumor immune responses,which can include optimal timing,dosing or combinations.Objective:To comprehensively investigate the characteristics and clinical significance of the changes of circulating immune cells during definitive（chemo）radiotherapy[（chemo）RT]in cervical cancer patients with the aim to guide individualized treatment.Methods:Retrospectively analyzing the clinical data of cervical cancer patients treated with definitive（chemo）RT,and single cell transcriptome sequencing（scRNAseq）of peripheral blood mononuclear cells（PBMCs）before and during（chemo）RT was prospectively analyzed.Results:Five main results are listed below.（1）The characteristics and influencing factors of bone marrow suppression during concurrent chemoradiotherapy（CCRT）for patients with cervical cancerFrom January 2015 to June 2018,a total of 60 cervical cancer patients with International Federation of Obstetrics and Gynecology（FIGO）IB1-IVA and CCRT were analyzed.The incidence of grade 3-4 leukopenia,neutropenia,anemia and grade 2-4 thrombocytopenia during CCRT were 53.3%,35.0%,20.0%and 33.3%,respectively.The earliest time of severe myelosuppression occurred at the 3rd week.Myelosuppression was correlated with pre-treatment hemoglobin and platelet,but not associated with the pelvic and vertebral dose volume histogram（DVH）parameters.Myelosuppression during CCRT did not affect RT but reduced the dose of concurrent chemotherapy（P=0.009）.（2）The neutrophil nadirs（ANC nadir）and lymphocytes nadirs（ALC nadir）during CCRT have different prognostic effects in cervical cancerFrom January 2015 to September 2019,a total of 87 cervical cancer patients with FIGO IB1-IVA and CCRT were analyzed.The cutoff values predicting overall survival（OS）of ANC nadir and ALC nadir were determined by time-dependent receiver operating characteristic（ROC）curve.Compared with ANC nadir ≤2.14×109/L（N=76）,patients with ANC nadir>2.14×109/L（N=11）had lower 2-year OS（54.5%vs 88.9%,P=0.035）.Compared with ALC nadir>0.2×109/L（N=49）,patients with ALC nadir≤0.2×109/L（N=38）had lower 2-year OS（75.3%vs 90.8%,P=0.008）.Multivariate analysis showed that both ANC nadir and ALC nadir were independently associated with OS.Patients with ANC>5.19×109/L before CCRT were more likely to occur ANC nadir>2.14×109/L during CCRT and patients with ALC<2.05×109/L before CCRT was more likely to occur ALC nadir≤0.2×109/L during CCRT.（3）Early onset of severe lymphopenia（EOSL）during pelvic（chemo）RT correlated with mean body dose（MBD）and predicted poor survival in cervical cancerFrom January 2015 to December 2019,a total of 104 cervical cancer patients with FIGO IB1-IVA and pelvic（chemo）RT were analyzed.EOSL was defined as first onset of grade 3-4 lymphopenia ≤3 weeks from the start of RT.MBD was the mean radiation dose absorbed by the body during the whole course of EBRT.The percentage of patients with EOSL was 59.6%.MBD（P=0.04）,concurrent cisplatin（P=0.011）,and pre-RT absolute lymphocyte count（ALC）（P=0.001）were associated with EOSL.There was a dose-response relationship between MBD and risk of EOSL.Patients with EOSL had decreased OS（2-year 75.1%vs 91.1%,P=0.021）and progression-free survival（PFS）（2-year 71.2%vs 83.7%,P=0.071）.The Cox model for predicting OS including EOSL and MBD has a C index of 0.835 and an area under the curve（AUC）of 0.872.A nomogram was established to predict 2-year OS,which can distinguish patients with different prognosis.（4）A machine learning model for grade 4（G4）lymphopenia prediction during pelvic（chemo）RT in patients with cervical cancerA total of 130 cervical cancer patients treated with pelvic（chemo）RT were included.Forty-three（33.1%）patients had G4 lymphopenia during（chemo）RT.On multivariate analysis,G4 lymphopenia was associated with poor OS（Hazard ratio[HR]3.91,95%confidence interval[CI]1.34-11.38,P=0.01）.Seven significant factors（Eastern Cooperative Oncology Group[ECOG]performance score,pre-RT hemoglobin,pre-RT lymphocytes,concurrent chemotherapy,gross tumor volume of regional lymphadenopathy[GTV_N volume],body volume,and maximum dose of planning target volume receiving at least 55Gy[PTV₅500 Dmax]）were obtained by Elastic-net logistic regression models and were included in the final prediction model for G4 lymphopenia.The model’s predicting ability was validated in the completely independent test set（AUC=0.8,accuracy=0.79）.（5）scRNA-seq of PBMCs for cervical cancer patients treated with CCRTTwo cervical cancer patients treated with CCRT were enrolled.Before and 3-4 weeks after the start of CCRT,PBMCs were isolated and scRNA-seq was performed.A total of 35379 cells were obtained.All cells were classified into 19 cell subtypes,including B cell,CD1C+CLEC10A+DC,CD4+T.naive,CD4+Tem,CD8+T.cytotoxic,CD8+T.naive,CD8+Tem,CD14+Monocyte,CD16+Monocyte,Erythrocyte,MAIT,MDSC,Macrophage,Megakaryocyte,NK,Plasma cell,Progenitor-like cell,Tfh,and Treg.After CCRT,the percentage and absolute count of CD4+T.naive,CD8+T.cytotoxic,CD8+T.naive,CD8+Tem,Macrophage,NK,and B cells decreased;the percentage and absolute count of CD4+Tem,MDSC,progenitor-like cell,Tfh,Treg,CD14+Monocytes,and CD16+Monocytes increased.30 genes were up-regulated and 3 genes were down-regulated in CD8+T cells after CCRT.Gene Ontology（GO）enrichment analysis showed that 30 up-regulated genes were mainly enriched into apoptosis pathway and positive regulation pathway of inflammatory response;three down-regulated genes were mainly enriched in the negative regulation of mitogen-activated protein kinase（MAPK）cascade pathway.Only one down regulated gene（TRBV25-1）showed significant differential expression between tumor and normal tissue and prognostic significance for OS in the Cancer Genome Atlas（TCGA）and the Genotype-Tissue Expression（GTEx）cervical cancer datasets.With regard to 5 immune checkpoint molecules（PD1,CTLA4,TIGIT,LAG3,and HAVCR2[TIM3]）and 2 immunosuppressive molecules（IDO1 and TGFB1）in CD8+T cells,after CCRT the expression of LAG3,IDO1,and TGFB1 increased,the expression of TIGIT decreased,and the expression of PDCD1,CTLA4,and HAVCR2 were not significantly changed.Among the 7 genes,only TIGIT showed significant differential expression between tumor and normal tissue and prognostic significance for OS in TCGA and GTEx cervical cancer datasets.Conclusions:Circulating immune cells change significanfly during definitive（chemo）RT and are prognostic factors for survival in patients with cervical cancer.It is worth further studying their clinical values as biomarkers.