Alterations In Glycosylation Of Saliva Glycoprotein From Patients With Early-stage Breast Cancer

Background:Breast cancer is the common female malignant tumor in worldwide with a high morbidity.To date,there are no ideal biomarkers for the detection of early-stage breast cancer.During the process of tumorigenesis and development,the glycosylation of proteins from tissue and body fluids are altered.The saliva is an ideal,non-invasive source for clinical diagnosis,and cost-effective saliva test has developed beyond oral cancer to other solid tumors.Thus,the hypothesis that shown the salivary protein glycosylation related with benign or malignant breast tumour,as potential biomarkers,have discriminatory power for the detection of breast cancer was investigated in this study.Method:Firstly,90 salivary samples from patients with breast benign diseases(BB,n=30),new diagnoses of I stage breast cancer patients,n=30),and ? stage breast cancer patients(BC-?,n=30),as well as 30 age-matched healthy volunteers(HV)were mixed to probe the difference of salivary glycopatterns with development of breast disease using lectin microarrays and blotting analysis.Then 266 saliva samples were divided into retrospective cohort(HV=36,BB=35,BC-?=36,BC-?=32)and validation cohort(HV=31,BB=30,BC-?=30,BC-?=36).Based on the data of lectin microarray from retrospective cohort,the candidate lectins were elected to distinguish different types of salivary samples and to construct the diagnostic models of early-stage breast cancer by a logistic stepwise regression.Further,the performance of the diagnostic models were assessed by receiver operating characteristic(ROC)curve analysis in the validation cohort.Result:1.According to the microarray data of salivary mixtures,the normalized fluorescent intensities(NFIs)of 11 lectins showed the significant differences among four types of pooled saliva.Among them,5 lectins(DBA,PNA,PHA-E+L,UEA-? and PWM)exhibited significantly differences in at least one breast tumour group compare with HV,but the NFIs of these lectins were not significantly different among breast disease saliva mixtures.The total abundance of ?GalNAc,GalNAc?l-3(Fuc?l-2)Gal,Gal?l-3GalNAc?,bisecting GlcNAc,multiple-antennary complex-type N-glycan,and Fuc?l-2Gal?l-4Glc(NAc)showed significant changes in saliva from patients with breast tumour.Moreover,the NFIs of 6 lectins(MAL-I,ECA,NPA,BPL,PTL-? and BS-?)significantly increased or decreased among three breast tumour groups.The results expressed the total abundance of Gal?l-4/3GlcNAc,High-Man,Man?l-6Man,Gal?l-3GalNAca-Ser/Thr and Gal?l-3/6Gal/Glc were altered in saliva for the female with benign tumour or cancer.The lectin blotting test based on the 4 selected lectins(PHA-E+L,NPA,MAL-? and BS-?)validate the reliability of variation patterns of lectin microarray in four saliva groups.2.Depended on the array data of retrospective cohort,8 out of the previous 11 lectins(PNA,PHA-E+L,UEA-I,PWM,MAL-?,NPA,BS-? and PTL-?)and PHA-E were selected as candidates to distinguish the four types salivary samples.However,the ROC analysis showed unsatisfactory diagnostic capacity for single lectins,except for PHA-E+L and PWM in HV/breast diseases patients(area under the ROC curve>0.70).Here,the four lectin-based models(Model BD,Model BB,Model BC-?,as well as Model BC-?)was constructed to differentiate the different saliva samples.The constructive models in the retrospective cohort were then applied to the validation cohort,in order to evaluate the diagnostic power.The result shows:79 cases of 96 BD and 26 cases of 31 HV were correctly classified by Model BD(AUC:0.902,sensitivity:0.823,specificity:0.839);23 cases of 30 BB and 23 cases of 30 BC-I as well as 25 cases of 36 BC-? were correctly classified by Model BB(AUC:0.796,sensitivity:0.727,specificity:0.767);21 cases of 30 BC-? as well as 57 case of 66 samples from other two groups were correctly classified by Model BC-?(AUC:0.781,sensitivity:0.700,specificity:0.864);finally,31 cases of 36 BC-? and 33 case of 60 salivary samples of other two groups were correctly classified by Model BC-?(AUC:0.759,sensitivity:0.861,specificity:0.550).In a word,four constructive diagnostic models achieved high diagnostic powers with an AUC value greater than 0.750 in new cohort,which suggest the anomalous protein glycopatterns in saliva as potential biomarkers may contribute to the screening for patients with early-stage breast cancer.