Construction Of A Model For Predicting Preoperative Lymph Node Metastasis Of Oral Squamous Cell Carcinoma Based On Exosomal CircRNA

ObjectiveTo explore the influencing factors of lymph node metastasis in patients with oral squamous cell carcinoma(OSCC),and to screen and verify the exosome circular RNA(circ RNA)related to lymph node metastasis of OSCC and to establish a model for predicting the risk of preoperative lymph node metastasis.Methods1.The distribution of cervical lymph node metastasis in 506 patients with OSCC was analyzed retrospectively.According to the proportion of 3:1,the patients were randomly divided into the modeling group(n=387)and the validation group(n=119).Unconditional logistic regression model was used to evaluate and calculate odds ratio(OR)and its 95% confidence interval(CI)between the influencing factors in the modeling group and the risk of lymph node metastasis in OSCC.2.According to the results of multivariate analysis,the influencing factors of lymph node metastasis of OSCC in the model group were selected to construct the nomogram model to predict the risk of lymph node metastasis before operation,and the prediction performance of nomogram model was evaluated in the modeling group,and the internal verification was carried out in the validation group.3.Four samples of preoperative plasma of OSCC with lymph node metastasis,4 samples of preoperative plasma of OSCC without lymph node metastasis and 4samples of postoperative plasma of OSCC with lymph node metastasis were selected for circ RNAs microarray detection.Then we demonstrated the difference of the expression of circ RNA between the patients of OSCC with lymph node metastasis before and after surgical treatment,OSCC with metastasis and without metastasis.The intersection of exosomal circ RNA of the three comparison group is used as the results of the initial screening.4.Circ RNAs was screened from plasma exocrine bodies of 77 patients with OSCC by real-time fluorescence quantitative PCR(q RT-PCR),and the relationship between the expression level of circ RNAs and the risk of lymph node metastasis of OSCC was studied.Finally,the circ RNA which was significantly related to the lymph node metastasis of OSCC and the clinical features that have influence on the lymph node metastasis are selected to construct the Circ RNA-based nomogram model.Results1.The overall lymph node metastasis rate of OSCC patients was 40.12%.Cervical lymph node metastasis mainly occurred in levels Ⅰ,Ⅱ and Ⅲ,of which levelⅡ had the highest lymph node metastasis rate(62.50%),while levels Ⅳ and Ⅴ were less involved.2.The results of multivariate analysis in the modeling group showed that patients with squamous cell carcinoma with differentiated degree of biopsy had a higher risk of lymph node metastasis than patients with squamous intraepithelial lesions(OR=2.62,95% CI: 1.38-4.98).The risk of lymph node metastasis in patients with buccal carcinoma and suboral carcinoma was significantly higher than that in gingival carcinoma(OR=3.32,95% CI: 1.20-9.20,OR=3.80,95% CI: 1.13-12.80),MRI reported that patients with positive lymph nodes had a higher risk of lymph node metastasis(OR=7.39,95% CI: 3.55-15.40).In addition,the risk of lymph node metastasis in patients with the largest tumor diameter ≥ 2.6cm OSCC reported by MRI was 2.13 times higher than that in patients with < 2.6cm(95% CI: 1.26-3.60).There was a positive correlation between low white blood cell level and the risk of lymph node metastasis in patients with OSCC(OR=5.46,95% CI: 1.48-20.09).3.Based on the variables selected from the multivariate analysis of the modeling group,a Nomogram model was constructed to predict the risk of preoperative lymph node metastasis.The results of ROC and DCA showed that Nomogram model had better predictive performance and higher clinical application value than MRI.4.The results of microarray showed that there were 7 plasma exosomal circ RNA from tumor tissue and related to lymph node metastasis,of which 2 were up-regulated and 5 down-regulated.Through the screening of the expression abundance of circ RNA microarray and the effect of primer design,it was found that the following three exosomes with the most obvious differences were used for further analysis: hsa＿circ RNA＿047733,hsa＿circ RNA＿024144 and hsa＿circ RNA＿403472.5.qRT-PCR showed that the expression level of hsa＿circ RNA＿047733 in OSCC lymph node metastasis positive group was lower than that in lymph node metastasis negative group.Hsa＿circ RNA＿047733 with higher expression level could reduce the risk of lymph node metastasis in OSCC.6.The Circ RNA-based nomogram model was superior to the Nomogram model based on patients’ clinical characteristics in terms of differentiation,calibration,goodness-of-fit and clinical application value.Conclusions1.The prediction factors of lymph node metastasis in patients with OSCC were the degree of differentiation of biopsy,the maximum diameter of tumor and the status of lymph nodes reported by MRI,tumor subsite and the level of white blood cells.The Nomogram model constructed in this study has a good performance in predicting the risk of lymph node metastasis.2.High expression of hsa＿circ RNA＿047733 could reduce the risk of lymph node metastasis in OSCC.The inclusion of exosomal hsa＿circ RNA＿047733 on the basis of Nomogram model based on patients’ clinical characteristics could effectively improve the ability of nomogram model to predict the risk of lymph node metastasis.