Development Of Methods For Differential Diagnosis Of Patients With Elevated Serum CA125Level

Ovarian cancer is the most lethal gynecological malignancy, causing144,000deaths worldwide per year (http://apps.who.int/ghodata/). Most patients have reached an advanced stage at the time of diagnosis. This may partly attributed to its vague and nonspecific symptoms. CA125is the most commonly used serum biomarker for diagnosis of epithelial ovarian cancer (EOC). Elevation of its serum levels was observed in most of non-mucinous EOC. The sensitivity of CA125for detection of EOC is about80%. However, its specificity is relatively low. Some patients with benign gynecological disease such as endometriosis, or non-ovarian malignancies, or even apparently healthy women may have elevated serum CA125level. It is reported that in patients with endometriosis, which also usually present with an adnexal mass and affect more than10%of women in reproductive age, up to27%have abnormal CA125levels. These pose significant confusion to diagnosis of EOC.CA125is a highly glycosylated mucin encoded by MUC16gene. In healthy women, it is synthesized mainly by mesothelial cells. Irritations to mesothelium, such as ascetic fluid, pelvic inflammatory disease, are common causes of elevated serum CA125levels. However, in EOC, cancerous synthesis and secretion is the main source of serum CA125. 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, it is reasonable to expect a distinct glycosylation pattern in EOC-derived CA125, which may serve as a biomarker to improve the diagnostic accuracy of EOC. In searching for this, we performed antibody-lectin sandwich assay using serum from patients with EOC, or benign adnexal mass.In part one, we established antibody-lectin ELISA assay for detection of glycosylation pattern of CA125. Firstly, to diminish background signals induced by glycans attached on Fc fragment of antibody, we derivatized these glycan with NaIO4and MPBH. After derivatization, background signals for most lectins decreased significantly. Then, we preincubated lectins with their corresponding inhibiting sugar before appling them to antibody-lectin ELISA assay. Most lectin binding with antigen were inhibited in a sugar concentration dependent manner, which suggested that testing signals were induced by a lectin-glycan binding. To prove that lectins bound with glycans attached to CA125, we incorporated CA125into CA125free serum to create serum of different concentration of CA125. Five antibody-lectin assay showed linear relationship with concentration of CA125.In part â…¡, we tested CA125glycosylation pattern using those five antibody-lectin assays. Among them, VVA, which recognizes Tn antigen, atchieved AUC of0.83in discriminating malignancy-derived CA125from benign condition-derived CA125. When cutoff value was set to be0.2, its sensitivity and specificity were82.5%and74.5%. Further more, we applied CA125/Tn ELISA in differentiating diagnosis of26untreated patients with CA125>100U/ml. Its positive predict value is30%, while its negative predict value is93.8%.In conclusion, CA125/Tn ELISA is a useful and reliable method for differentiating diagnosis of patients with elevated serum CA125level.