| CA125 (cancer antigen 125), encoded by the MUC16 gene, is a highly glycosylated protein which is the most widely used biomarker in gynecology tumors. CA125 may be elevated in a number of benign conditions, such as pregnancy, endometriosis, uterus myoma or gynecological inflammatory condition, resulting in high false positive rate and unnecessary mental burden.Under different health condition, CA125 can be synthesized by various kinds of cells, such as mesothelial cells and cancer cells. It is well known that obvious changes occur in glycosylation machinery in malignant tissues, which result in aberrant glycan pattern in glycoproteins synthesized by these tissues. Thus, we can infer that distinct glycosylation pattern in CA125 probably serves as a biomarker to distinguish the benign conditions from the malignant conditions with elevated CA125.Containing 22,152 amino acids, CA125 is a complex high molecular O-glycosylated protein. Overexpression of Ga1β1-3Ga1NAca (TF or T antigen) is a typical change in tumor cells. Investigations suggest that T antigen increases in about 90% of all human cancers, including ovary cancer. Accordingly, we wonder if T antigen could potentially aid in the differentiation of benign from malignant gynecological conditions among those people with elevated CA125.Mass spectrometry glycoprofiling is a common method to detect CA125 aberrant glycosylation, but not commonly used in hospital. Besides, some researchers noticed that the lectins are highly specific for carbohydrate moieties, and developed some lectin-based glycomic approaches. For example in 2007, an efficientmethod for analyzing variation in glycosylation in complex biological samples was developed by Songming Chen et al. They used antibody microarray to capture the multiple proteins followed by detection of glycans with lectins. Chemical derivatization was taken to block lectin binding to glycan groups on Fc fragment of the spotted antibodies, which guaranteed the binding between lectin and the captured antigen. Detected once the lectin is biotinylated, absorbance could suggest the quantity of glycans on the captured antigen. Considering peanut agglutinin, derived from the fruits of Arachis hypogaea, binds preferentially to the T-antigen, we referred to the method developed by Songming Chen et al. and established CA125/T ELISA assay in order to provide a new method for differential diagnosis for patients with elevated CA125.In part one, we established CA125/T ELISA assay for detection of T antigen of CA125. Firstly, to diminish background signals induced by glycans attached on Fc fragment of antibody, we derivatized these glycans with NaIO4 and MPBH. After derivatization, background signals for PNA decreased. Then, we preincubated PNA with its corresponding inhibiting sugar before appling them to antibody-lectin ELISA assay.PNA binding with antigen was inhibited in a sugar concentration dependent manner, which suggested that testing signals were induced by a lectin-glycan binding. Moreover, in order to prove that PNA bound with glycans attached to CA125 instead of other glycoproteins in serum, we incorporated CA125 into CA125 free serum to prepare samples with concentration gradient of CA125. Consequently, the PNA binding ability was linear with the concentration of CA125.In part two, we collected 186 patients with elevated CA125, including 73 malignant cases, 11 borderline cases and 102 benign cases, from Obstetrics and Gynecology Hospital. Expression of T antigen on CA125 detected by CA125/T Elisa Assay was significantly higher in malignant conditions than in benign conditions (p<0.001). The CA125/T Elisa Assay resulted in high diagnostic power in differentiating benign conditions from malignant conditions in gynecological patients with elevated CA125 (ROC AUC 0.824, at 64.3% sensitivity and 91.2% specificity). Prospective study showed the positive predictive value was 54.5% and the negative predictive value was 91.2%. No significant relationship was observed between T antigen expression and grade.We also found menstrual status and age could not interfere in detection.In part three, we found that patients in T antigen high expression group had a poorer prognosis compared with lower expression group.By COX risk regression analysis, T antigen over expression in CA125 affected the PFS significantly.In Conclusion,the CA125/T Elisa Assay may be a potential diagnostic tool to predict tumor malignancy in gynecological patients with elevated CA125. |
