Effect Of External Beam Radiation Therapy For Cervical Cancer On The Microbiome Of Patients And Its Relationship With Radiosensitivity

Objective:A retrospective cohort study was conducted to compare the effects of external beam radiation therapy(EBRT)on the microbiome of 36 patients with primary cervical cancer treated with radiation therapy,to explore the correlation between the microbiome characteristics and the efficacy of EBRT in cervical cancer,and to provide new insights into the prognostic targets for cervical cancer.Methods:1.Enrolment and multi-site microbial collection:tumour tissues,vaginal secretions,stools,urine,and oral swabs from primary cervical cancer patients aged between 18 and 75 years who attended the Third Affiliated Hospital of Kunming Medical University between December 2021 and July 2022 were collected before and at the end of 25 external irradiation radiotherapy sessions.All study participants signed an informed consent form.2.Evaluation of the effectiveness of EBRT and collection of clinical data:a pre-radiotherapy tumor volume(pre-RT TV)was measured using MRI or CT of the pelvis in all patients prior to EBRT.After 25 EBRT sessions,pelvic MRI was performed again to measure the post external beam radiation therapy tumor volume(post-EBRT TV),and the tumor volume reduction rate(TVRR)was calculated by[(pre-RT TV-post-EBRT TV)/pre-RT TV×100].Patient information such as age,ethnicity,pathological stage and classification were collected,and extracted from the electronic medical record system.3.Next-generation sequencing(NGS)of DNA from multi-site microbial samples:a total of 36 patients,including tumor tissue,vaginal secretions,stools,urine,and oral swab specimens were included in this study for a total of 349 cases.The Illumina platform was used for sequencing the“V3-V4”region of microbial DNA in all samples.Alpha diversity analysis,Beta diversity analysis,and VENN analysis were performed in“R”,and the stacked bar plot of the community composition,microbial diversity box plots,and the receiver operating characteristic curve(ROC)were plotted.Linear Discriminant Analysis Effect Size(LEf Se)was performed by LEf Se software.The Wilcoxon rank sum test was used to characterize the multi-site flora before radiotherapy and the changing trend of the flora after radiotherapy.Using TVRR=66.7%after external radiation radiotherapy as the optimal cut-off value,patients with TVRR≥66.7%were in the objective response(OR)group and those with TVRR<66.7%were in the inferior response(IR)group,comparing the differences in microbial diversity and composition between the two groups and screening for differences microbiota.Combined with the Kyoto Encyclopedia of Genes and Genomes signaling pathway database,the metabolic pathway differences between the OR group and the IR group tumor microbiome were compared using“R”to create a heat map of metabolic differences.4.Immunohistochemical staining:Immunohistochemical staining was performed to compare the difference in immune infiltration of CD8~+T cells or granzyme B(Gzm B)within the tumour tissue of the OR and IR groups before radiation treatment,combined with screening for differential microorganisms for Spearman correlation analysis.Results:1.After EBRT,microbialα-diversity decreased in the tumor tissue and vagina and increased in the gut,urethra,and oral cavity,with significant differences inβ-diversity at these sites.2.Before EBRT,there was a significant difference in the composition of the intra-tumor microbiome between the OR and IR groups,with a lower baseline alpha-diversity of tumor microorganisms in the OR group than in the IR group and a negative correlation with TVRR,in which patients achieved a better outcome with EBRT.LEf Se analysis revealed significantly higher abundances of Bifidobacteriaceae,Beijerinckiaceae,and Orbaceae within the OR group than the IR group at the family level.3.The density of baseline CD8~+T-cell and Gzm B~+infiltration in the OR group was higher than that in the IR group.Spearman’s correlation analysis revealed that the baseline Bifidobacteriaceae enrichment was significantly and positively correlated with the density of CD8~+T(P=0.010)and Gzm B~+(P=0.012)infiltration within the tumor tissue.The ACE index was significantly and negatively correlated with the infiltration density of CD8+T(P=0.009)and Gzm B+(P=0.038).These results suggest that the low diversity of the tumor microbiome and the enrichment of Bifidobacteriaceae may be involved in the anti-tumor response by promoting the recruitment and activation of CD8~+T cells.4.The area under curve(AUC)of the ROC plotted by combining the absolute abundances of Bifidobacteriaceae,Beijerinckiaceae,and Orbaceae was 0.823.5.The comparison of differences in KEGG metabolic pathways revealed that the IR group tumor microbiome was significantly more metabolic than the OR group at all levels of metabolic pathways.Conclusions:1.Radiotherapy can significantly alter the diversity and composition of the multi-site microbiome of patients with cervical cancer.2.The lower microbial diversity and higher abundance of Bifidobacteriaceae in the tumor prior to radiotherapy may facilitate the recruitment and activation of CD8+T cells,resulting in better EBRT outcomes for patients.3.The absolute abundance of Bifidobacteriaceae,Beijerinckiaceae and Orbaceae may have potential as biomarkers for predicting the efficacy of EBRT in cervical cancer.