The Association Between Neoadjuvant Chemoradiotherapy And Gut Microbes And Systemic Immunoinflammatory Markers In Rectal Cancer

BackgroundRectal cancer is the malignant tumor which has the fourth morbidity and the fifth lethality in China,seriously threatening people's health.Preoperative neoadjuvant concurrent chemoradiotherapy(NCCRT)combined with radical rectal resection is the standard treatment for locally advanced rectal cancer(LARC),but the pathological complete remisson(pCR)rate is just about 15%-27%.If the patients with complete pathological remission can be screened out effectively to enter the "Wait and Watch" mode,the lesions and over-treatment caused by the surgery can be avoided to a great extent.Hence,how to find a non-invasive marker that preferably predicts the efficacy of NCCRT has become a major problem in clinical practiceAs a research hotspot in recent years,the microbiome has shown superior clinical prospects both in fecal microbiota transplantation(FMT)and probiotic treatment.The balance of the human-microbes' symbiotic ecosystem is extremely pivotal for maintaining human homeostasis and health.When the balance was disturbed,many studies related to microbes' disorders and tumors,cardiovascular diseases,cognitive diseases,psychotic diseases and immunoinflammatory diseases have already been reported.It is even believed that colorectal cancer is not only a genetic disease,but also a microbial disease.The intestinal microbes participate in the regulation of the oncogenesis,progression and therapeutic effect of gastrointestinal tumors by producing metabolites and toxins,affecting cell proliferation,modulating inflammation and immunity,and inducing DNA damage.However,there has been no such comprehensive research reported about NCCRT's modulation of microbes and microbes' effect on chemoradiotherapy sensitivity yet.Radiotherapy can also exert anti-tumor effects by regulating immunity in addition to inducing direct DNA damage,it will be conducive to deeply understand the action mode of NCCRT by explore the regulation of NCCRT on systemic immune and inflammation related markersBased on analysis above,we collected fecal samples before,during and after NCCRT treatment separately and also fresh tumor tissue samples,para-cancerous tissue samples in subsequent surgery in LARC patients for 16s rRNA pyrosequencing to explore the remodeling of NCCRT on the microbes,and also to screen out the differential genus between patients with variant response to NCCRT so as to provide a basis for microbes and chemoradiotherapy sensitivity.Peripheral blood samples of patients were also collected at three simultaneous sampling time points above.We detected the expression level of soluble type of immunomodulatory proteins as CTLA4,PD1,PD-L1,LAG3 and also analyzed the variation tendency of systemic inflammatory indexes such as Platelets/Lymphocytes Ratio(PLR),Neutrophils/Lymphocytes Ratio(NLR),Systemic Immune-inflammation Index(SII).Then these indexes' relationships with the efficacy in NCCRT were further investigated.At the same time,we examined the density of TILs of different subtypes in tumor biopsy tissue and surgical tumor tissue after NCCRT treatment.We also conducted an exploratory analysis of differential genera' correlation with immunological and inflammatory indexes mentioned above to provide a basis for subsequent clinical and basic researchPart I Neoadjuvant chemoradiotherapy remodels intestinal microbes affecting the Objective:efficacy in LARCTo explore the remodeling effect of NCCRT on intestinal and intratumor microbes;To screen out the differential genus of patients with variant efficacy and provide a basis for studying the microbes and chemoradiotherapy sensitivity so as to find a novel biomarker that better predicts the efficacy of NCCRT.Materials and Methods:A total of 39 LARC patients who underwent standard NCCRT were enrolled.The fecal samples before,during and after NCCRT treatment respectively and also fresh tumor tissue samples,para-cancerous tissue samples in subsequent surgery were collected in succession for 16s rRNA V4 pyrosequencing to identify the remodeling effect of NCCRT on intestinal and intratumor microbes,and then screened out the differential genus between patients with variant efficacy through bioinformatics analysis.Results:1.The diversity of intestinal microbes decreased significantly during the NCCRT compared to baseline before NCCRT(p=0.001),and the tumor tissue had similar diversity to the para-cancerous tissue2.Patients with better NCCRT efficacy had a higher intestinal abundance as a whole during the neoadjuvant treatment(p=0.0347).3.Firmicutes/Bacteroidetes ratio gradually decreased throughout NCCRT at the phylum level.And Fusobacterium,Parvimonas,Clostridium sensu stricto showed a significant decrease after the NCCRT at the genus level(p<0.001).The tumor tissue was similar to the adjacent tissues in bacteria composition4.After the completion of NCCRT,the abundance of Clostridium sensu stricto.Fusobacterium,Ruminococcus,Clostridium IV,Oscillibacter,Clostridium_X1Vb,Faecalibacterium and Enterobacter in patients of good efficacy was significantly lower than ones with poor efficacy(p<0.05)5.In patients of good efficacy,the abundance of Ruminococcus,Clostridium_?,Oscillibacter,Clostridium_XlVb,Faecalibacterium and Enterobacter was significantly decreased throughout NCCRT(p<0.05)while moderate increase was observed in ones of poor efficacy.Conclusions:1.NCCRT decreased the gut microbes' diversity markedly and changes the composition and abundance of intestinal microbes,remodeling the gut microbes2.Patients with better NCCRT efficacy generally had higher intestinal flora abundance.3.Several genera were related to the efficacy NCCRT after the treatment endedNCCRT showed differential shaping of intestinal bacteria among patients with different efficacy.Eight genera belonged to Clostridia,Fusobacteria and Gammaproteobacteria may affect the efficacy of NCCRT.Part ? Modulation of systemic immune inflammatory markers throughout NCCRT Objective:in rectal cancer and its correlation with gut microbesTo investigate the variation tendency of systemic inflammatory indexes PLR,NLR,SII and soluble immunomodulatory proteins sCTLA4,sPD1,sPD-L1 and sLAG3 throughout the NCCRT processes in LARC,and to generalize differential typical features of patients with variant efficacy,to compare the infiltrating characteristics of TILs in both biopsy and surgical tumor tissue,to make an exploratory analysis of differential intestinal genera with systemic immunological and inflammatory indexes aboveMaterials and Methods:Thirty-nine patients with LARC who underwent NCCRT were enrolled.We collected three separate peripheral blood samples before,during and after the NCCRT for detecting the expression level of soluble immunomodulatory proteins sCTLA4,sPD1,sPD-L1 and sLAG3.We calculated systemic inflammatory markers PLR,NLR,SII based on blood routine test.We also compared local TILs infiltration characteristics before and after NCCRT treatment by immunohistochemical staining.Statistical analysis was conducted to summarize the NCCRT's effect on systemic immunological and inflammatory indexes and generalize the differential typical features of patients with variant efficacy.We made an exploratory demonstration on the correlation of screened differential intestinal genera with systemic immunological and inflammatory indexes via bioinformatics exploratory analysis.Results:1.The systemic inflammatory indexes PLR,NLR,and SII were significantly increased in the first half(p<0.001)and kept stable in the latter half of NCCRT processes2.There was no obvious discrepancy of PLR,NLR,and SII and the same variation trend was observed in patients with different efficacy3.The secretion of circulating sCTLA4,sPD1,sPDL1,and sLAG3 decreased significantly in the first half of NCCRT(p<0.001)and increased to the baseline level in the second half.4.There was no obvious discrepancy of the expression level of sCTLA4,sPD1,sPDL1,and sLAG3 but different variation tendency in patients with different efficacy.Patients demonstrating a significant decrease in the first half of NCCRT had much better efficacy.5.The variation tendency of sCTLA4,sPD1,sPDL1,and sLAG3 in patients with good efficacy was observed in unison with the whole enrolled patients,a significant decrease in the first half of NCCRT was observed(p<0.001),meanwhile the expression levels stayed stable in the poor efficacy group6.The expressions of PD-1,PD-L1 and Foxp3 in tumor tissue specimens after NCCRT were lower compared to those in tumor biopsy specimens before NCCRT.PD-1 was highly expressed in patients with better NCCRT efficacy and Foxp3 was higher in patients with poor efficacy.Nevertheless,statistical power was weakened because of limited number of samples,the above trend did not reach statistically significant difference7.Correlation analysis showed that circulating CTLA4,PD1,LAG3 were positively correlated with the abundance of Fusobacterium,and negatively correlated with the abundance of Faecialbacterium,Alistipes and Ruminococcus2Conclusions:1.The NCCRT process increased the systemic inflammatory level of LARC patients.A significant increase of PLR,NLR,SII in the first half and a stable status in the second half of NCCRT were demonstrated both in patients with good efficacy and bad efficacy.2.Patients with significant decrease of soluble immunoregulatory proteins sCTLA4,sPD1,sPDL1,and sLAG3 in the first half seemed more sensitive to NCCRT3.There were correlations between circulating immunomodulatory proteins and some intestinal genera in the NCCRT treatment.Circulating sCTLA4,sPD1,sLAG3 were positively correlated with the abundance of Fusobacterium,and negatively correlated with the abundance of Faecialbacterium,Alistipes and Ruminococcus2The innovation and limitation of this subject Innovations of the subject1.For the first time,this study compared the gut microbes between LARC patients with different efficacy and explored the differential genus associated with efficacy,providing a basis for microbes and chemoradiotherapy sensitivity and finding non-invasive clinical markers.2.This study elucidated the changes of the gut microbes' diversity,composition and abundance in LARC patients by dynamically monitoring the gut microbiome throughout the NCCRT processes and provided evidence for the remodeling effect of the microbes by NCCRT.3.This study researched the dynamic changes of systemic inflammatory indexes and circulating immunomodulatory proteins throughout NCCRT in LARC patients and summarized their change characteristics of patients with variant efficacy,deepening the understanding of the action mode of NCCRT.We also made an exploratory analysis of the correlation between differential genus and peripheral immunological markers in NCCRTLimitations of the subject1.Although this study was a dynamic monitoring design throughout the NCCRT treatment,the number of enrolled patients was relatively small.An expansion of sample size for further verification will be needed2.There was only one single evaluation criteria(p CR)of NCCRT efficacy at short term but no more recurrence or survival data,a long-term follow-up of enrolled patients is still needed to be done next3.This study only explored the correlation between differential gut genera and specific peripheral immunological indexes while the mechanism was not discussed in depth.We should establish animal models and conduct cell experiments to further verify the mechanism how the selected differential genus affected the efficacy of NCCRT.