EOCa and GOCa are two different types of ovarian cancer that affect women of different age groups. While EOCa is more common in pre-menopausal women, GOCa is predominantly a cancer that affects adolescents and young women under 30 years of age. EOCa arises from the epithelium or outer cells covering the ovary and it accounts for more than 90% of ovarian cancer cases. GOCa differs from EOCa in that it arises from the cells that mature into eggs that are formed inside the ovary .
By using 2-DE and image analysis, we have demonstrated the alteration of serum high abundance proteins expression involving higher expression of CLU, AAT, AATf, LRG, HAP and HAPc in sera of both cohorts of patients with EOCa and GOCa. However, the expression of ACT was only enhanced in EOCa patients while patients with GOCa demonstrated lower levels of AHS and enhanced expression of CPL compared to the negative controls. Since the controls used in the present study were serum samples obtained from normal healthy individuals, we cannot totally exclude the possibility that the aberrant protein expression may be due to inflammation of the ovary rather than cancer. Nevertheless, similar preliminary experiments performed on patients with inflammation of the ovary indicated that the high abundance acute-phase protein expression was markedly different from those of the EOCa and GOCa patients (data not shown).
The elevated levels of CLU in both cohorts of EOCa and GOCa patients and enhanced expression of ACT only in patients with EOCa were confirmed by ELISA. ELISA also confirmed the up-regulated expression of HAP in EOCa patients but was not able to demonstrate the difference in the expression of HAP and CPL in patients with GOCa as well as the up-regulated expression of AAT in both cohorts of the ovarian carcinoma patients. CLU and CPL were positively-detected in the biopsy samples of all patients tested while staining of ACT was positive in 13/17 patients' tissues analyzed.
CLU is implicated in diverse physiological functions including the regulation of cell growth and survival . Its enhanced expression has been described in breast cancer , hepatocellular carcinoma , desmoplastic melanoma , colorectal cancer , bladder cancer , lymphomas  as well as ovarian cancer . Multiple isoforms of CLU, arise from various post-translational modification processes, have been detected . In the present study, two groups of CLU were detected in the 2-DE protein profiles. While the main group of CLU was up-regulated in the sera of EOCa and GOCa patients, CLU2 was not abnormally expressed. These results are compatible with the recent findings of Rodriguez-Pineiro and coworkers in their studies on colorectal cancer . Our data also emphasized the expression of CLU in the malignant tissues of all ovarian carcinoma patients analyzed.
Like CLU, CPL is another protein reported to be elevated in sera of patients with diverse types of cancer. Elevated levels of CPL has been described in studies on thyroid cancer , melanoma , lymphoma , kidney and urinary tract cancer , gastrointestinal cancer , cervical cancer [9, 25], uterine cancer [9, 26] and nasopharyngeal carcinoma . When bound to copper, CPL induces angiogenesis, which supports growth of tissues and tumors . In the present study, detection of CPL in tissues of all patients tested is indicative of the strong association of angiogenesis with malignancy. The up-regulated expression of CPL was also significantly detected by 2-DE in the GOCa patients but not in patients with EOCa.
ACT, a member of the serine proteinase inhibitor family, is an acute-phase response protein . Its association with cancer is rather restrictive unlike CLU and CPL. Previous studies have shown that the levels of ACT were also elevated in patients with breast cancer [7, 29], and the excessive ACT was likely to be synthesized by the breast epithelial cells [30, 31]. Hence, ACT may be also synthesized by the epithelial cells of the ovary which explains its observed up-regulated expression in sera of patients with EOCA but not GOCa patients both by using 2-DE and ELISA. In the tissues of the ovarian cancer patients, however, ACT was positively-stained in both the two biopsy samples of GOCa patients tested as well as 76% of the biopsy samples of EOCa patients.
The enhanced levels of AAT in sera of ovarian carcinoma patients that were detected in our 2-DE studies were not supported by the results of our ELISA experiments. Nevertheless, the up-regulated expression of AAT, which was always linked to liver diseases , as well as its N-terminal fragment, has been previously reported in the sera of patients with cancers of the prostate, lung and breast  although this association was not well understood. Similarly, our 2-DE experiments also detected the up-regulated expression of AATf, which were likely to be generated by the proteolysis of AAT, in both EOCa and GOCa patients. This appears to indicate that AAT was actively processed within the cancer microenvironment. AAT was also positively-detected in 59% biopsy samples of patients analyzed, inclusive of both subtypes of ovarian carcinoma.
Like AAT, ELISA was not able to confirm the different altered expression of AHS and HAP in sera of ovarian carcinoma patients by 2-DE studies. Our previous reports have indicated that the 2-DE proteomic approach is more sensitive than ELISA with many of the proteins analysed showed more than ten-fold difference in sensitivity between the two techniques [7, 8]. One possibility for the difference in sensitivity as well as the discrepancy in the results is that the up-regulated proteins may have triggered the humoral immune response in patients and caused binding of the patients' antibodies to the serum proteins. Such binding would certainly interfere with the ELISA and reduce its sensitivity.
The correlation between HAP expression and ovarian carcinoma has been widely reported [34, 35]. Studies have also shown both the α- and β-subunits of HAP to be significantly increased in early stage ovarian carcinoma patients [36, 37]. In the case of AHS, our data showed down-regulation of the protein in sera of both cohorts of EOCa and GOCa patients. The low concentration of AHS in ovarian carcinoma serum may be associated with depressed cellular immunity  and short-term mortality of patients similar to that reported for patients with liver cancer .
Due to the lack of antiserum, we were not able to validate the results of the 2-DE experiments on overexpression of LRG in sera and tissues of EOCa and GOCa patients. LRG, a secretory protein of unknown function , has been noted for its acute-phase protein characteristics . We have also recently reported the up-regulated expression of LRG in sera of patients with endometrial adenocarcinoma .
Our present data obtained from patients with ovarian carcinoma, when taken together with our previous reported studies on sera of patients with cancers of the breast , nasopharynx , cervix and endometrium , demonstrated that the cancer patients were aberrant in the expression of their serum high abundance acute-phase proteins, and this was simultaneously detected using the gel-based proteomic analysis on whole human serum samples without the need to deplete albumin or immunoglobulins. Equally intriguing is that the different types of cancer that were studied so far appear to demonstrate distinctive altered patterns of serum high abundance protein expression. In the case of ovarian carcinoma, this explicitly involved elevated levels of CLU in both the EOCa and GOCa patients and enhanced expression of ACT only in patients with EOCa.
The different altered expression of selective high abundance acute-phase serum proteins of patients with EOCa and GOCa suggests that they may be used as protein signatures that may facilitate diagnoses and/or monitoring of the cancers. Ovarian carcinoma is usually asymptomatic in its early stages and development. For most patients, the disease is often widespread at the time of diagnosis, and this is also due to the absence of sensitive and reliable serological markers. CA125, the currently accepted serum marker for diagnosis of ovarian carcinoma, is limited in sensitivity as it is detected in about 50% of patients at stage 1 and 75–90% of women with advanced stage of the disease. Moreover, it lacks specificity as it is also elevated in 26% of non-ovarian malignancies, 14% benign ovarian disease and 9% of benign gynecologic disorders . Thus the need for more specific and efficient biomarkers for diagnosis of ovarian carcinoma is pressing. Although limited in scale, identification of the link between selective aberrantly expressed serum proteins with EOCa and GOCa as demonstrated in the present gel-based proteomic study is a necessary prelude to the conduct of a larger scale clinical investigation in pursuit of additional diagnostic markers that are more reliable and sensitive.