In this report, we confirmed that seven previously reported blood biomarkers (CA125, MIF-1, leptin, prolactin, OPN, IGF-II, and p53 AAbs) are associated with the presence of ovarian cancer [19
]. We further demonstrated that among them, seropositivity of p53 AAbs and elevated MIF are associated with type II, not type I ovarian cancer; while the other five are not type-specific markers for ovarian cancer, although the blood levels of CA125 and IGF-II exhibit more dramatic change in type II than type I tumor patients when compared to healthy controls (). The specificity of p53 AAbs for type II tumors supports the notion that ‘ovarian cancer’ is not a single disease, requiring different biomarkers be utilized for each specific disease type. Similarly, NY-ESO-1 AAbs were not apparent in the plasma of type I ovarian cancer patients, but they were detected in a small fraction of type II ovarian cancer patients. Notably, type II tumors are more aggressive and prevalent than type I tumors and need blood biomarkers to aid the early detection [10
], therefore it is reasonable to focus on type II tumors for the identification of biomarkers in the future.
In theory, the efficient combination of biomarkers should consist of biomarkers that are not correlated with each other because the combined effect of such markers will be closer to an additive effect. Through the statistical analysis of currently promising candidate markers, our study suggests that seropositivity of p53 AAbs might be a useful blood marker to combine with CA125 for the early detection of type II ovarian cancer. This notion was supported by the low level of correlation between the production of p53 AAbs and CA125 in type II ovarian cancer patients (), and by the comparable seropositivity of p53 AAbs in the patients with negative and positive CA125 (). These data clearly indicated the additional benefit of combining p53 AAbs with CA125 over CA125 alone in the detection of type II ovarian cancer. Since CA125 positivity correlates with increased ovarian cancer volume measured under laparoscopy/laparotomy [19
], it suggests that the production of p53 AAbs by type II ovarian cancer patients is relatively independent of the extent of disease. A similar observation was made for NY-ESO-1 antibodies, suggesting that AAbs can be triggered by low volume type II ovarian cancer. Indeed, p53 AAbs were previously shown to be present in sera of patients with asbestosis several years prior to a diagnosis of cancer [26
], therefore it is highly possible that immune system recognizes limited amount of mutant p53 accumulation occurring early in the development of ovarian cancer [12
] and mount a well-adapted humoral immune response by generating detectable levels of p53 AAbs early enough when other markers are not detectable [33
]. We found that although the magnitude of the p53 AAb responses tended to be lower in CA125 negative patients, the percentage of p53 AAb seropositivity tended to be higher in this group of patients when we used a cut-off providing 100% specificity for p53 AAbs. However, we recognize that this result is only based on our small cohort study; and that much more work is needed in the future to evaluate how useful it is to combine p53 AAbs with CA125 in the early detection of type II ovarian cancer.
We found that recently identified markers MIF, leptin, prolactin, OPN, and IGF-II [30
] exhibited differential levels in the plasma of type II ovarian cancer patients compared to disease-free controls (); however, none of them behaved as well as CA125 as a single marker in our sample set based on AUC of ROC curve (). We observed a similar trend when we compared all the ovarian cancer patients with disease-free controls (data not shown). Similar to CA125, leptin, prolactin, OPN, and IGF-II also exhibited differential blood levels in patients with type I ovarian cancer and disease-free controls (), which may partially explain their association with ovarian cancers (i.e. presumably type I and type II), especially low stage disease, reported before [30
] since low stage disease is typically skewed more to type I tumors than high stage cases.
Previous studies [32
] identified significant seropositivity of IgGs against several other tumor associated antigens including NY-ESO-1, ALPP, CTSD, B23, GRP78, SSX in ovarian cancer patients, but not in normal controls or benign tumor patients. We did not detect a differential antibody response against ALPP, CTSD, B23, GRP78, and SSX with our patient cohort, but this may reflect the antigen source used in our immunoassay. Taylor et al
used tumor-derived exosomal proteins in their assays, whereas we used recombinant tagged proteins expressed in 293TT cells and captured from detergent lysates. When Taylor et al
compared the exosomal proteins and their recombinant counterparts produced in bacteria, the exosomal proteins exhibited much stronger immunoreactivity with the patient-derived antibodies [32
], suggesting the importance of secondary modifications in AAb recognition of these antigens that are apparently not produced by over-expression in 293TT cells. Conversely, we have successfully used p53 and NY-ESO-1 expressed in 293TT cells or in bacteria to detect AAbs in ovarian cancer patients (data not shown).
In summary, our data suggests seropositivity of p53 AAbs as a biomarker relatively independent of CA125 for type II, but not type I, epithelial ovarian cancer. It is particularly useful in CA125 negative patients and combination of p53 AAbs and CA125 effectively improved the discrimination ability to detect type II ovarian cancer. Our results are based on comparison between plasma from women with and without clinically confirmed disease and should be extended to blood specimens from asymptomatic patients. Thus, combining the detection of p53 AAbs and CA125 in screening for type II ovarian cancer among an asymptomatic population warrants further study, but is unlikely to be helpful to identify patients with type I ovarian cancer. Further, the utility of p53 AAbs, CA125 and IGF-II as markers to discriminate between type II and type I in patients with low stage disease also needs further examination in independent and prospective studies.