In this study, we provide evidence that the use of immunodepletion to remove highly abundant proteins from serum followed by DIGE analysis can be used to quantitate protein levels in serum from ovarian cancer patients and non-cancer controls for the discovery of candidate biomarkers. By these techniques, we identified nine proteins present at higher levels in ovarian cancer sera than in control sera.
Several of the proteins identified in this study, for example, haptoglobin [26
] and inter-alpha globulin inhibitor H4 [31
], have previously been shown to be increased in the sera of ovarian cancer patients by proteomic and other techniques, validating our screen for potential biomarkers in serum. Haptoglobin was first shown to be elevated in the sera of women with ovarian cancer almost 40 years ago [28
]. This finding has subsequently been confirmed numerous times, most recently by 2-DE [26
], SELDI, and ELISA [29
]. Haptoglobin is one of the highly abundant proteins targeted by the immunodepletion columns, and was visibly depleted from the non-cancer serum (, arrow) by the MARS column. Visual examination of the MARS-fractionated sera by silver stained 2-DE gels revealed increased levels of haptoglobin in the cancer serum pool in both the high abundance (, arrow) and low abundance (, arrow) fractions. We conclude that the MARS column effectively depleted haptoglobin from serum except when present at elevated levels, such as in sera from ovarian cancer patients, in which case the column depletion capacity is insufficient for complete removal of the protein.
We also identified several less well-studied proteins that were elevated in ovarian cancer sera and have the potential to serve as biomarkers. For example, LRG1 was expressed at increased levels in ovarian cancer sera by DIGE, and subsequently was validated by Western blot (), and by ELISA in individual, undepleted serum samples (manuscript in preparation). Although LRG1 has been classified as an “acute phase” protein [32
], we have evidence that LRG1 is also produced by ovarian cancer cells and may contribute to the increased levels of LRG1 found in patient sera (manuscript in preparation). Others have found by 2-DE, that specific isoforms of LRG1 have increased expression in the proliferative endometrium of women undergoing fertility treatment who subsequently became pregnant independent of treatment [34
], and in the peritoneal fluid of women with uterine leiomyomas [35
]. Interestingly, Ferrero and colleagues also saw a significant correlation between leiomyoma size and LRG1 expression [35
]. We identified LRG1 peptides in DIGE spots representing four different isoforms, although only one spot was significantly increased in ovarian cancer sera, and only a single protein band of ~51 kDa was detected by Western blot. Taken together, these data indicate a potential role for at least one isoform of LRG1 in the physiology of the female reproductive organs, and a potential biomarker for ovarian cancer.
We also found that ficolin 3 was more than 3-fold elevated in ovarian cancer sera by DIGE analysis, and its relative expression was validated by Western blot. Although ficolin 3 is thought to play a role in immune activation [36
], it has been identified by MS in ovarian cancer ascites cells [38
] suggesting a potential role in ovarian cancer, as well. Ficolin 3 has also been identified in a DIGE analysis of serum in prostate cancer progression [20
]. Thus, ficolin 3 and LRG1 both warrant further investigation into their expression in ovarian cancer cells and their specificity as potential biomarkers for ovarian cancer.
We also validated the level of serum expression for alpha-1 antichymotrypsin, an acute phase protein that may play a role in ovarian cancer by binding and regulating members of the kallikrein family of serine proteases [40
]. Alpha-1 antichymotrypsin was detected as a protein of ~ 56-65 kDa that was visibly increased in the cancer serum pools by Western blotting (). In the DIGE experiments, six spots were identified as AACT ( and ), with an average 3-fold increase in the cancer serum pools (ranging from a 2.25 to 4.9-fold increase in the cancer sera), which is corroborated by the Western blotting results ().
Several of the proteins found to be increased in ovarian cancer serum relative to control samples were acute phase proteins that can be elevated in response to infection or injury [32
], and as such may not be specific to ovarian cancer. Nevertheless, in a recent analysis of 204 serum markers on a large cohort of sera from women with ovarian cancer as well as benign gynecological disease, several acute phase and inflammatory proteins were among the best discriminators of malignancy [41
]. These findings support the notion that inflammation plays a role in cancer initiation and progression [42
] and indicate that acute phase proteins may prove to be critical in the development of a robust “multi-analyte” panel of ovarian cancer biomarkers.
Others have used post-translational modifications as a method to detect and enrich for biomarkers in serum of ovarian cancer patients. Ogata and colleagues used immunodepletion and 2-DE to analyze post-translationally modified proteins by means of specific gel staining methods [43
]. They identified a phosphorylated isoform of fibrinogen alpha that was upregulated in ovarian cancer patients. Furthermore, Jackson et al. [44
] used DIGE and 2-D lectin profiling for the discovery of glycoprotein biomarkers. They found the albumin-related protein, afamin, was present at decreased levels in ovarian cancer compared to control sera.
Immunodepletion of serum followed by DIGE analysis has been used for biomarker discovery in a number of other cancer types including prostate [20
], pancreatic [18
], and lung cancers [16
]. Similar to our results in ovarian cancer, serum levels of haptoglobin, inter-alpha globulin inhibitor H4, and LRG1 were elevated in the medium and low abundance serum proteins of lung and pancreatic cancers [16
]. Taken together, these results suggest that these proteins may be indicative of a general response to cancer, rather than tumor-specific proteins; although in the cases of haptoglobin and LRG1, there is evidence that these proteins are synthesized by ovarian cancer cells [30
For biomarker discovery, we used 60 serum samples from patients with primarily stage III and stage IV serous ovarian carcinoma and 60 serum samples from female non-cancer “controls” that were randomly pooled into six groups of ten patient samples. The samples were pooled to reduce variation between individual samples and to increase the overall number of samples tested. We selected patients with advanced disease under the assumption that these patients may have higher levels of ovarian cancer proteins in their sera than patients with early stage disease, thus increasing the likelihood of detecting ovarian cancer specific biomarkers. There is some evidence that the serum protein profile may change with disease progression [45
], which may limit the utility of late stage samples for the discovery of early stage biomarkers. However, a recent analysis of 21 tumor biomarkers in a large cohort of patients demonstrated consistent biomarker expression across stages within a particular ovarian cancer subtype, suggesting that differential expression of biomarkers is related to ovarian cancer subtype and not stage [47
]. Regardless, it will be important to validate the candidate biomarkers identified using a large cohort of individual serum samples, which are age-matched and include samples from women with early stage cancers and benign gynecological conditions, as well as healthy controls. Moreover, the candidate biomarkers detected herein may prove useful for the detection of disease recurrence.