Identification of Multiple Novel Protein Biomarkers Shed by Human Serous Ovarian Tumors into the Blood of Immunocompromised Mice and Verified in Patient Sera

The most cancer-specific biomarkers in blood are likely to be proteins shed directly by the tumor rather than less specific inflammatory or other host responses. The use of xenograft mouse models together with in-depth proteome analysis for identification of human proteins in the mouse blood is an under-utilized strategy that can clearly identify proteins shed by the tumor. In the current study, 268 human proteins shed into mouse blood from human OVCAR-3 serous tumors were identified based upon human vs. mouse species differences using a four-dimensional plasma proteome fractionation strategy. A multi-step prioritization and verification strategy was subsequently developed to efficiently select some of the most promising biomarkers from this large number of candidates. A key step was parallel analysis of human proteins detected in the tumor supernatant, because substantially greater sequence coverage for many of the human proteins initially detected in the xenograft mouse plasma confirmed assignments as tumor-derived human proteins. Verification of candidate biomarkers in patient sera was facilitated by in-depth, label-free quantitative comparisons of serum pools from patients with ovarian cancer and benign ovarian tumors. The only proteins that advanced to multiple reaction monitoring (MRM) assay development were those that exhibited increases in ovarian cancer patients compared with benign tumor controls. MRM assays were facilely developed for all 11 novel biomarker candidates selected by this process and analysis of larger pools of patient sera suggested that all 11 proteins are promising candidate biomarkers that should be further evaluated on individual patient blood samples.     

A serum glycomics approach to breast cancer biomarkers

Because the glycosylation of proteins is known to change in tumor cells during the development of breast cancer, a glycomics approach is used here to find relevant biomarkers of breast cancer. These glycosylation changes are known to correlate with increasing tumor burden and poor prognosis. Current antibody-based immunochemical tests for cancer biomarkers of ovarian (CA125), breast (CA27.29 or CA15-3), pancreatic, gastric, colonic, and carcinoma (CA19-9) target highly glycosylated mucin proteins. However, these tests lack the specificity and sensitivity for use in early detection. This glycomics approach to find glycan biomarkers of breast cancer involves chemically cleaving oligosaccharides (glycans) from glycosylated proteins that are shed or secreted by breast cancer tumor cell lines. The resulting free glycan species are analyzed by MALDI-FT-ICR MS. Further structural analysis of the glycans can be performed in FTMS through the use of tandem mass spectrometry with infrared multiphoton dissociation. Glycan profiles were generated for each cell line and compared. These methods were then used to analyze sera obtained from a mouse model of breast cancer and a small number of serum samples obtained from human patients diagnosed with breast cancer or patients with no known history of breast cancer. In addition to the glycosylation changes detected in mice as mouse mammary tumors developed, glycosylation profiles were found to be sufficiently different to distinguish patients with cancer from those without. Although the small number of patient samples analyzed so far is inadequate to make any legitimate claims at this time, these promising but very preliminary results suggest that glycan profiles may contain distinct glycan biomarkers that may correspond to glycan "signatures of cancer."     

Integrated Proteomic Analysis of Human Cancer Cells and Plasma from Tumor Bearing Mice for Ovarian Cancer Biomarker Discovery

Background

The complexity of the human plasma proteome represents a substantial challenge for biomarker discovery. Proteomic analysis of genetically engineered mouse models of cancer and isolated cancer cells and cell lines provide alternative methods for identification of potential cancer markers that would be detectable in human blood using sensitive assays. The goal of this work is to evaluate the utility of an integrative strategy using these two approaches for biomarker discovery.

Methodology/Principal Findings

We investigated a strategy that combined quantitative plasma proteomics of an ovarian cancer mouse model with analysis of proteins secreted or shed by human ovarian cancer cells. Of 106 plasma proteins identified with increased levels in tumor bearing mice, 58 were also secreted or shed from ovarian cancer cells. The remainder consisted primarily of host-response proteins. Of 25 proteins identified in the study that were assayed, 8 mostly secreted proteins common to mouse plasma and human cancer cells were significantly upregulated in a set of plasmas from ovarian cancer patients. Five of the eight proteins were confirmed to be upregulated in a second independent set of ovarian cancer plasmas, including in early stage disease.

Conclusions/Significance

Integrated proteomic analysis of cancer mouse models and human cancer cell populations provides an effective approach to identify potential circulating protein biomarkers.     

Elevated activity of cytidine 5'-monophospho-N-acetylneuraminic acid hydrolase in serum of ovarian cancer patients as a possible indicator of malignancy.

Cytidine 5′-monophospho-N-acetylneuraminic acid hydrolase activity has been demonstrated in homogenates of normal ovary and ovarian epithelial adenocarcinomas, as well as in the sera of normal individuals and ovarian cancer patients. The specific activity of the enzyme in ovarian tumors is significantly reduced compared to normal ovaries. In pre-operative sera of these patients, the activity is elevated about 2 fold in comparison with age and blood group matched controls. The patients with lower tissue enzyme activity have higher serum values. After tumor reductive surgery, serum levels of this enzyme are diminished, but are still higher than controls. Clinical use of this assay for detection and management of ovarian neoplasia is promising.     

Peritoneal fluids from patients with certain gynecologic tumor contain elevated levels of bioactive lysophospholipase D activity

Levels of lysophosphatidic acid (LPA), an important phospholipid mediator, in serum and ascitic fluid from ovarian cancer patients were shown to be higher than those from healthy women and from patients with other type of cancer, respectively. Although LPA in human serum seems mainly to be generated by lysophospholipase D (lysoPLD), the source and pathway for LPA in the ascitic fluid remain still obscure. In this study, we examined whether lysoPLD activity producing bioactive LPA in human peritoneal fluid was significantly elevated under pathological statuses. Lysophospholipase D activity in human peritoneal fluids was measured by quantifying choline released from exogenous lysophosphatidylcholine on their incubation at 37 degrees C. We also compared the activity of lysoPLD in sera from patients with different gynecologic diseases. We found relatively high lysoPLD activity in peritoneal fluids from patients with ovarian cancer, dermoid cyst or mucinous cystadenoma, whereas there were no significant differences in the serum lysoPLD activity among clinical groups and healthy subjects. The lysoPLD in the peritoneal fluid was found to have similar substrate specificity and metal ion requirement to those of serum lysoPLD, that has been identified as autotaxin, a tumor cell-motility stimulating protein. Our results suggest that increased lysoPLD activity in peritoneal fluid from patients with certain gynecologic tumors might be relevant to its potential of tumor progression.     

Evaluation of proteomics-identified CCL18 and CXCL1 as circulating tumor markers for differential diagnosis between ovarian carcinomas and benign pelvic masses

A lack of sensitive and specific tumor markers for early diagnosis and treatment is a major cause for the high mortality rate of ovarian cancer. The purpose of this study was to identify potential proteomics-based biomarkers useful for the differential diagnosis between ovarian cancer and benign pelvic masses. Serum samples from 41 patients with ovarian cancer, 32 patients with benign pelvic masses, and 41 healthy female blood donors were examined, and proteomic profiling of the samples was assessed by surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectroscopy (MS). A confirmatory study was also conducted with serum specimens from 58 patients with ovarian carcinoma, 37 patients with benign pelvic masses, and 48 healthy women. A classification tree was established using Biomarker Pattern Software. Six differentially expressed proteins (APP, CA 125, CCL18, CXCL1, IL-8, and ITIH4) were separated by high-performance liquid chromatography and identified by matrix-assisted laser desorption/ionization (MALDI)-MS/MS and database searches. Two of the proteins overexpressed in ovarian cancer patients, chemokine CC2 motif ligand 18 (CCL18) and chemokine CXC motif ligand 1 (CXCL1), were automatically selected in a multivariate predictive model. These two protein biomarkers were then validated and evaluated by enzyme-linked immunosorbent assay (ELISA) in 535 serum specimens (130 ovarian cancer, 64 benign ovarian masses, 36 lung cancer, 60 gastric cancer, 55 nasopharyngeal carcinoma, 48 hepatocellular carcinoma, and 142 healthy women). The combined use of CCL18 and CXCL1 as biomarkers for ovarian cancer had a sensitivity of 92% and a specificity of 97%. The multivariate ELISA analysis of the two putative markers in combination with CA 125 resulted in a sensitivity of 99% for healthy women and 94% for benign pelvic masses, and a specificity of 92% for both groups; these values were significantly higher than those obtained with CA 125 alone (p and lt;0.05). We conclude that serum CCL18 and CXCL1 are potentially useful as novel circulating tumor markers for the differential diagnosis between ovarian cancer and benign ovarian masses.     

Use of an immunoaffinity-mass spectrometry-based approach for the quantification of protein biomarkers from serum samples of lung cancer patients

It is a challenging task to verify and quantify potential biomarkers expressed at elevated levels in sera from cancer patients. An immunoaffinity-mass spectrometry-based approach has been developed using antibodies to enrich proteins of interest from sera followed by mass spectrometry-based quantification. Antibodies specific to the protein of interest were immobilized to hydrazide resin via the carbohydrate moiety on the Fc region of the antibody. Captured proteins were eluted, reduced, alkylated, and digested with trypsin. Peptides were analyzed by LC coupled with multiple reaction monitoring approach, and quantification was achieved by the addition of stable isotope-labeled (heavy) standard peptides. Using this methodology, we were able to achieve a linear response from 15 to 250 ng/ml for carcinoembryonic antigen (CEA), a known tumor biomarker. Moreover we observed elevated levels of CEA in sera samples from lung cancer patients that to our knowledge is the first time that circulating CEA has been detected by mass spectrometry-based analysis. This approach was further applied to potential protein biomarkers discovered from tumor cell lines and tumor tissues. A linear response was obtained from a multiplex spiking experiment in normal human sera for secretory leukocyte peptidase inhibitor (4-500 ng/ml), tissue factor pathway inhibitor (TFPI) (42-1000 ng/ml), tissue factor pathway inhibitor 2 (TFPI2) (2-250 ng/ml), and metalloproteinase inhibitor 1 (TIMP1) (430-1000 ng/ml). A replicate experiment for a single concentration value yielded a relative coefficient of variation better than 11% for TFPI, secretory leukocyte peptidase inhibitor, and TFPI2. The expression level of the proteins in lung cancer patient sera was assayed by an immunoaffinity-multiple reaction monitoring method, and the results were comparable with those obtained from ELISA. This immunoaffinity-mass spectrometry-based quantification approach thus provides a specific and accurate assay for verifying the expression of potential biomarkers in patient serum samples especially for those proteins for which the necessary reagents for ELISA development are unavailable.     

Integrated pipeline for mass spectrometry-based discovery and confirmation of biomarkers demonstrated in a mouse model of breast cancer

Despite their potential to impact diagnosis and treatment of cancer, few protein biomarkers are in clinical use. Biomarker discovery is plagued with difficulties ranging from technological (inability to globally interrogate proteomes) to biological (genetic and environmental differences among patients and their tumors). We urgently need paradigms for biomarker discovery. To minimize biological variation and facilitate testing of proteomic approaches, we employed a mouse model of breast cancer. Specifically, we performed LC-MS/MS of tumor and normal mammary tissue from a conditional HER2/Neu-driven mouse model of breast cancer, identifying 6758 peptides representing >700 proteins. We developed a novel statistical approach (SASPECT) for prioritizing proteins differentially represented in LC-MS/MS datasets and identified proteins over- or under-represented in tumors. Using a combination of antibody-based approaches and multiple reaction monitoring-mass spectrometry (MRM-MS), we confirmed the overproduction of multiple proteins at the tissue level, identified fibulin-2 as a plasma biomarker, and extensively characterized osteopontin as a plasma biomarker capable of early disease detection in the mouse. Our results show that a staged pipeline employing shotgun-based comparative proteomics for biomarker discovery and multiple reaction monitoring for confirmation of biomarker candidates is capable of finding novel tissue and plasma biomarkers in a mouse model of breast cancer. Furthermore, the approach can be extended to find biomarkers relevant to human disease.     

An Outlook on Ovarian Cancer and Borderline Ovarian Tumors: Focus on Genomic and Proteomic Findings

Among the gynaecological malignancies, ovarian cancer is one of the neoplastic forms with the poorest prognosis and with the bad overall and disease-free survival rates than other gynaecological cancers. Ovarian tumors can be classified on the basis of the cells of origin in epithelial, stromal and germ cell tumors. Epithelial ovarian tumors display great histological heterogeneity and can be further subdivided into benign, intermediate or borderline, and invasive tumors. Several studies on ovarian tumors, have focused on the identification of both diagnostic and prognostic markers for applications in clinical practice. High-throughput technologies have accelerated the process of biomolecular study and genomic discovery; unfortunately, validity of these should be still demonstrated by extensive researches on sensibility and sensitivity of ovarian cancer novel biomarkers, determining whether gene profiling and proteomics could help differentiate between patients with metastatic ovarian cancer and primary ovarian carcinomas, and their potential impact on management. Therefore, considerable interest lies in identifying molecular and protein biomarkers and indicators to guide treatment decisions and clinical follow up. In this review, the current state of knowledge about the genoproteomic and potential clinical value of gene expression profiling in ovarian cancer and ovarian borderline tumors is discussed, focusing on three main areas: distinguishing normal ovarian tissue from ovarian cancers and borderline tumors, identifying different genotypes of ovarian tissue and identifying proteins linked to cancer or tumor development. By these targets, authors focus on the use of novel molecules, developed on the proteomics and genomics researches, as potential protein biomarkers in the management of ovarian cancer or borderline tumor, overlooking on current state of the art and on future perspectives of researches.Key Words: Ovarian cancer, borderline ovarian tumors, markers, genomics, proteomics, oncogenes.     

Precise mapping of increased sialylation pattern and the expression of acute phase proteins accompanying murine tumor progression in BALB/c mouse by integrated sera proteomics and glycomics

Inbred BALB/c mouse implanted with murine tumors serves as an attractive model system for the studies of cancer biology in immuno-competent individuals. It is anticipated that tumor progression would induce notable pathophysiological consequences, some of which manifested as alteration in serum proteomic and glycomic profiles. Similar to sera derived from human cancer patients and immuno-compromised mice bearing human tumors, we show in this work that BALB/c mice of the same genetic background but bearing two distinct tumor origins both exhibited elevated expression levels of acute phase proteins including haptoglobin and serum amyloid P protein, in response to tumor progression. Such common traits are generally not informative nor qualifying as biomarkers. Additional mass spectrometry (MS)-based glycomic mapping nevertheless detected distinctive changes of sialylation pattern on the complex type N-glycans. MALDI MS/MS sequencing afforded a facile but definitive identification of an increase in internal Neu5Gcalpha2-6 sialylation on the GlcNAc of the Neu5Gc2-3Gal1-3GlcNAc terminal sequence as a common feature whereas a substitution of Neu5Gc by Neu5Ac was found to be induced by colonic but not breast tumor. A more pronounced change was similarly detected on N-glycans derived from ascitic fluids representing late tumor progression stages. We next demonstrated that such distinct change in glycotope expression can be localized to a particular protein carrier by LC-MS/MS analysis of glycopeptides. Serotransferrin was identified as one such abundant serum glycoprotein, which changed significantly not in protein expression level but in terminal glycosylation pattern.     

Mass spectrometric identification of human prostate cancer-derived proteins in serum of xenograft-bearing mice

Lack of sensitivity and specificity of current tumor markers has intensified research efforts to find new biomarkers. The identification of potential tumor markers in human body fluids is hampered by large variability and complexity of both control and patient samples, laborious biochemical analyses, and the fact that the identified proteins are unlikely produced by the diseased cells but are due to secondary body defense mechanisms. In a new approach presented here, we eliminate these problems by performing proteomic analysis in a prostate cancer xenograft model in which human prostate cancer cells form a tumor in an immune-incompetent nude mouse. Using this concept, proteins present in mouse serum that can be identified as human will, by definition, originate from the human prostate cancer xenograft and might have potential diagnostic and prognostic value. Using one-dimensional gel electrophoresis, liquid chromatography, and mass spectrometry, we identified tumor-derived human nm23/nucleoside-diphosphate kinase (NME) in the serum of a nude mouse bearing the androgen-independent human prostate cancer xenograft PC339. NME is known to be involved in the metastatic potential of several tumor cells, including prostate cancer cells. Furthermore we identified six human enzymes involved in glycolysis (fructose-bisphosphate aldolase A, triose-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, alpha enolase, and lactate dehydrogenases A and B) in the serum of the tumor-bearing mice. The presence of human NME and glyceraldehyde-3-phosphate dehydrogenase in the serum of PC339-bearing mice was confirmed by Western blotting. Although the putative usefulness of these proteins in predicting prognosis of prostate cancer remains to be determined, the present data illustrate that our approach is a promising tool for the focused discovery of new prostate cancer biomarkers.     

循环肿瘤细胞在女性实体器官肿瘤中的研究进展

循环肿瘤细胞(circulating tumor cells,CTCs)指的是从实体的肿瘤或转移的病灶进入外周血液循环的恶性肿瘤细胞。自发现以来,随着其检验技术日趋成熟,循环肿瘤细胞(CTCs)日渐成为肿瘤学炙手可热的研究对象。因为它将通过外周血的检验来实现监测肿瘤的发生、发展、转移、复发等情况,相对于肿瘤实体活检,"液体活检"不仅让患者易于接受,更有利于医务工作者监测病情变化。本文综述了循环肿瘤细胞(CTCs)的检测方法并综述了循环肿瘤细胞在女性实体肿瘤--乳腺癌、卵巢癌、宫颈癌、子宫内膜癌中的研究进展。其中着重介绍了其在早期乳腺癌及复发转移性乳腺癌中的重大意义以及在评价治疗效果中的分子学特征。实践表明,循环肿瘤细胞(CTCs)与HE-4、CA125的联合应用在评估卵巢癌化疗敏感性中也具有重要的临床意义。     

Profiling of glycans in serum for the discovery of potential biomarkers for ovarian cancer

A glycomic approach is developed to identify oligosaccharide markers for ovarian cancer by rapidly profiling globally released oligosaccharides. Glycoproteins shed by cancer cells are found in the supernatant (or conditioned media) of cultured cells. In this approach, shed glycoproteins are profiled for their oligosaccharide content using beta-elimination conditions. Changes in glycosylation are monitored by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS). Because shed glycoproteins would also be found in serum, similar glycan profiling was performed to observe potential oligosaccharide markers. Oligosaccharide profiling data on a limited set of patient and normal serum samples were studied to determine potential glycan markers in ovarian cancer. We were able to demonstrate the presence of at least 15 unique serum glycan markers in all patients but absent in normal individuals. To determine the structure of the glycan biomarkers, a number of the ions were isolated and further analyzed using infrared multiphoton dissociation (IRMPD). One major advantage of this approach is that glycans are examined directly from patient sera without the need to obtain cancer biopsy specimens or to purify glycosylated proteins from these specimens.     

Shared immunoproteome for ovarian cancer diagnostics and immunotherapy: potential theranostic approach to cancer

Elimination of cancer through early detection and treatment is the ultimate goal of cancer research and is especially critical for ovarian and other forms of cancer typically diagnosed at very late stages that have very poor response rates. Proteomics has opened new avenues for the discovery of diagnostic and therapeutic targets. Immunoproteomics, which defines the subset of proteins involved in the immune response, holds considerable promise for providing a better understanding of the early-stage immune response to cancer as well as important insights into antigens that may be suitable for immunotherapy. Early administration of immunotherapeutic vaccines can potentially have profound effects on prevention of metastasis and may potentially cure through efficient and complete tumor elimination. We developed a mass-spectrometry-based method to identify novel autoantibody-based serum biomarkers for the early diagnosis of ovarian cancer that uses native tumor-associated proteins immunoprecipitated by autoantibodies from sera obtained from cancer patients and from cancer-free controls to identify autoantibody signatures that occur at high frequency only in cancer patient sera. Interestingly, we identified a subset of more than 50 autoantigens that were also processed and presented by MHC class I molecules on the surfaces of ovarian cancer cells and thus were common to the two immunological processes of humoral and cell-mediated immunity. These shared autoantigens were highly representative of families of proteins with roles in key processes in carcinogenesis and metastasis, such as cell cycle regulation, cell proliferation, apoptosis, tumor suppression, and cell adhesion. Autoantibodies appearing at the early stages of cancer suggest that this detectable immune response to the developing tumor can be exploited as early-stage biomarkers for the development of ovarian cancer diagnostics. Correspondingly, because the T-cell immune response depends on MHC class I processing and presentation of peptides, proteins that go through this pathway are potential candidates for the development of immunotherapeutics designed to activate a T-cell immune response to cancer. To the best of our knowledge, this is the first comprehensive study that identifies and categorizes proteins that are involved in both humoral and cell-mediated immunity against ovarian cancer, and it may have broad implications for the discovery and selection of theranostic molecular targets for cancer therapeutics and diagnostics in general.     

Clinical cancer proteomics: promises and pitfalls

Proteome analysis promises to be valuable for the identification of tissue and serum biomarkers associated with human malignancies. In addition, proteome technologies offer the opportunity to analyze protein expression profiles and to analyze the activity of signaling pathways. Many published proteomic studies of human tumor tissue are associated with weaknesses in tumor representativity, sample contamination by nontumor cells and serum proteins. Studies often include a moderate number of tumors which may not be representative of clinical materials. It is therefore very important that biomarkers identified by proteomics are validated in representative tumor materials by other techniques, such as immunohistochemistry. Proteome technologies can be used to identify disease markers in human serum. Tumor derived proteins are present at nanomolar to picomolar concentrations in cancer patient sera, 10(6)-10(9)-fold lower than albumin, and will give rise to correspondingly smaller spots/peaks in protein separations. This leads to the need to prefractionate serum samples before analysis. Despite various pitfalls, proteomic analysis is a promising approach to the identification of biomarkers, and for generation of protein expression profiles that can be analyzed by artificial learning methods for improved diagnosis of human malignancy. Recent advances in the field of proteomic analysis of human tumors are summarized in the present review.     

REG4 Is Highly Expressed in Mucinous Ovarian Cancer: A Potential Novel Serum Biomarker

Preoperative diagnostics of ovarian neoplasms rely on ultrasound imaging and the serum biomarkers CA125 and HE4. However, these markers may be elevated in non-neoplastic conditions and may fail to identify most non-serous epithelial cancer subtypes. The objective of this study was to identify histotype-specific serum biomarkers for mucinous ovarian cancer. The candidate genes with mucinous histotype specific expression profile were identified from publicly available gene-expression databases and further in silico data mining was performed utilizing the MediSapiens database. Candidate biomarker validation was done using qRT-PCR, western blotting and immunohistochemical staining of tumor tissue microarrays. The expression level of the candidate gene in serum was compared to the serum CA125 and HE4 levels in a patient cohort of prospectively collected advanced ovarian cancer. Database searches identified REG4 as a potential biomarker with specificity for the mucinous ovarian cancer subtype. The specific expression within epithelial ovarian tumors was further confirmed by mRNA analysis. Immunohistochemical staining of ovarian tumor tissue arrays showed distinctive cytoplasmic expression pattern only in mucinous carcinomas and suggested differential expression between benign and malignant mucinous neoplasms. Finally, an ELISA based serum biomarker assay demonstrated increased expression only in patients with mucinous ovarian cancer. This study identifies REG4 as a potential serum biomarker for histotype-specific detection of mucinous ovarian cancer and suggests serum REG4 measurement as a non-invasive diagnostic tool for postoperative follow-up of patients with mucinous ovarian cancer.     

Proteomic Profiling of a Mouse Model for Ovarian Granulosa Cell Tumor Identifies VCP as a Highly Sensitive Serum Tumor Marker in Several Human Cancers

The initial aim of this study was to identify novel serum diagnostic markers for the human ovarian granulosa cell tumor (GCT), a tumor that represents up to 5% of all ovarian cancers. To circumvent the paucity of human tissues available for analyses, we used the Ctnnb1(tm1Mmt/+);Pten(tm1Hwu/tmiHwu);Amhr2(tm3(cre)Bhr/+) transgenic mouse model, which features the constitutive activation of CTNNB1 signaling combined with the loss of Pten in granulosa cells and develops GCTs that mimic aggressive forms of the human disease. Proteomic profiling by mass spectrometry showed that vinculin, enolase 1, several heat shock proteins, and valosin containing protein (VCP) were more abundantly secreted by cultured mouse GCT cells compared to primary cultured GC. Among these proteins, only VCP was present in significantly increased levels in the preoperative serum of GCT cancer patients compared to normal subjects. To determine the specificity of VCP, serum levels were also measured in ovarian carcinoma, non-Hodgkin's lymphoma and breast, colon, pancreatic, lung, and prostate cancer patients. Increased serum VCP levels were observed in the majority of cancer cases, with the exception of patients with lung or prostate cancer. Moreover, serum VCP levels were increased in some GCT, ovarian carcinoma, breast cancer, and colon cancer patients who did not otherwise display increased levels of widely used serum tumor markers for their cancer type (e.g. inhibin A, inhibin B, CA125, CEA, or CA15.3). These results demonstrate the potential use of VCP as highly sensitive serum marker for GCT as well as several other human cancers.     

Tumor antigen NB/70K and CA 125 levels in the blood of preoperative ovarian cancer patients and controls: a preliminary report of the use of the NB12123 and CA 125 radioimmunoassays alone and in combination

The NB12123 and CA125 radioimmunoassays, murine monoclonal antibody assays for measuring circulating levels of human ovarian tumor associated antigens NB/70K and CA 125, respectively, have been previously described. In the present study, preoperative serum samples were obtained from patients undergoing laparotomy for benign neoplastic ovarian tumors (N = 16), cancer of the cervix (N = 22), cancer of the uterus (N = 20), and cancer of the ovary (N = 47). Controls (N = 50) were obtained from healthy blood bank donors. No correlation was observed between the levels of NB/70K and CA 125 in these samples (r2 = .079, linear regression analysis). In general, increasing levels of both antigens were present with increasing tumor burden and higher histological grade. In addition, both markers were most elevated in the serum of ovarian cancer patients with serous and unclassified adenocarcinomas. Using 40 AU and 35 unit cut-offs for the NB/70K and CA 125 assay, respectively, overall specificity for healthy controls and patients with benign diseases approaches 100%. The combined sensitivity of the assays for ovarian cancer patient sera in this study indicates that the assays may be helpful in establishing a pre operative diagnosis of ovarian cancer. Complementarity of the NB/70K and CA 125 assays has been demonstrated, indicating that one or both assays may be used to monitor as many as 85% of ovarian cancer patients.     

Serum S100A6 Concentration Predicts Peritoneal Tumor Burden in Mice with Epithelial Ovarian Cancer and Is Associated with Advanced Stage in Patients

Background

Ovarian cancer is the 5th leading cause of cancer related deaths in women. Five-year survival rates for early stage disease are greater than 94%, however most women are diagnosed in advanced stage with 5 year survival less than 28%. Improved means for early detection and reliable patient monitoring are needed to increase survival.

Methodology and Principal Findings

Applying mass spectrometry-based proteomics, we sought to elucidate an unanswered biomarker research question regarding ability to determine tumor burden detectable by an ovarian cancer biomarker protein emanating directly from the tumor cells. Since aggressive serous epithelial ovarian cancers account for most mortality, a xenograft model using human SKOV-3 serous ovarian cancer cells was established to model progression to disseminated carcinomatosis. Using a method for low molecular weight protein enrichment, followed by liquid chromatography and mass spectrometry analysis, a human-specific peptide sequence of S100A6 was identified in sera from mice with advanced-stage experimental ovarian carcinoma. S100A6 expression was documented in cancer xenografts as well as from ovarian cancer patient tissues. Longitudinal study revealed that serum S100A6 concentration is directly related to tumor burden predictions from an inverse regression calibration analysis of data obtained from a detergent-supplemented antigen capture immunoassay and whole-animal bioluminescent optical imaging. The result from the animal model was confirmed in human clinical material as S100A6 was found to be significantly elevated in the sera from women with advanced stage ovarian cancer compared to those with early stage disease.

Conclusions

S100A6 is expressed in ovarian and other cancer tissues, but has not been documented previously in ovarian cancer disease sera. S100A6 is found in serum in concentrations that correlate with experimental tumor burden and with clinical disease stage. The data signify that S100A6 may prove useful in detecting and/or monitoring ovarian cancer, when used in concert with other biomarkers.     

Genome-scale screen for DNA methylation-based detection markers for ovarian cancer

Background

The identification of sensitive biomarkers for the detection of ovarian cancer is of high clinical relevance for early detection and/or monitoring of disease recurrence. We developed a systematic multi-step biomarker discovery and verification strategy to identify candidate DNA methylation markers for the blood-based detection of ovarian cancer.

Methodology/Principal Findings

We used the Illumina Infinium platform to analyze the DNA methylation status of 27,578 CpG sites in 41 ovarian tumors. We employed a marker selection strategy that emphasized sensitivity by requiring consistency of methylation across tumors, while achieving specificity by excluding markers with methylation in control leukocyte or serum DNA. Our verification strategy involved testing the ability of identified markers to monitor disease burden in serially collected serum samples from ovarian cancer patients who had undergone surgical tumor resection compared to CA-125 levels.We identified one marker, IFFO1 promoter methylation (IFFO1-M), that is frequently methylated in ovarian tumors and that is rarely detected in the blood of normal controls. When tested in 127 serially collected sera from ovarian cancer patients, IFFO1-M showed post-resection kinetics significantly correlated with serum CA-125 measurements in six out of 16 patients.

Conclusions/Significance

We implemented an effective marker screening and verification strategy, leading to the identification of IFFO1-M as a blood-based candidate marker for sensitive detection of ovarian cancer. Serum levels of IFFO1-M displayed post-resection kinetics consistent with a reflection of disease burden. We anticipate that IFFO1-M and other candidate markers emerging from this marker development pipeline may provide disease detection capabilities that complement existing biomarkers.