Ovarian Cancer: In Search of Better Marker Systems Based on DNA Repair Defects

Ovarian cancer is the fifth most common female cancer in the Western world, and the deadliest gynecological malignancy. The overall poor prognosis for ovarian cancer patients is a consequence of aggressive biological behavior and a lack of adequate diagnostic tools for early detection. In fact, approximately 70% of all patients with epithelial ovarian cancer are diagnosed at advanced tumor stages. These facts highlight a significant clinical need for reliable and accurate detection methods for ovarian cancer, especially for patients at high risk. Because CA125 has not achieved satisfactory sensitivity and specificity in detecting ovarian cancer, numerous efforts, including those based on single and combined molecule detection and “omics” approaches, have been made to identify new biomarkers. Intriguingly, more than 10% of all ovarian cancer cases are of familial origin. BRCA1 and BRCA2 germline mutations are the most common genetic defects underlying hereditary ovarian cancer, which is why ovarian cancer risk assessment in developed countries, aside from pedigree analysis, relies on genetic testing of BRCA1 and BRCA2. Because not only BRCA1 and BRCA2 but also other susceptibility genes are tightly linked with ovarian cancer-specific DNA repair defects, another possible approach for defining susceptibility might be patient cell-based functional testing, a concept for which support came from a recent case-control study. This principle would be applicable to risk assessment and the prediction of responsiveness to conventional regimens involving platinum-based drugs and targeted therapies involving poly (ADP-ribose) polymerase (PARP) inhibitors.     

Methods for Identification of CA125 from Ovarian Cancer Ascites by High Resolution Mass Spectrometry

CA125 is the most widely used tumour marker in ovarian cancer with unsatisfactory sensitivity and specificity especially at early stage. It is quantified by antibody-based immunoassays; however different molecular weight isoforms have been described in the literature which have never been validated by mass spectrometry, potentially affecting the diagnostic accuracy and clinical reliability of the test. In this study, CA125 was detected by Western blot and its identity confirmed by mass spectrometry. Two-dimensional (2D) gel electrophoresis in combination with mass spectrometry revealed that positive Western blot signals up to 500 kDa are most likely false-positive interactions of M11-like and OC125-like antibodies. Fibronectin, identified as one of these false-positive interaction partners, increased the reading for CA125 in a first generation ELISA significantly (p = 0.02). The existence of low-molecular weight isoforms of CA125 is therefore questionable and is most likely reflecting cross-reactivity of the antibodies with other proteins. This would explain the conflicting reports on the molecular structure of CA125 and also the inconsistency of CA125 levels by different ELISAs. Our results are also the first steps towards a mass spectrometric assay for CA125 quantification, which would improve sensitivity and reliability.     

Deciphering the Molecular Nature of Ovarian Cancer Biomarker CA125

The ovarian cancer biomarker CA125 has been extensively investigated over the last 30 years. The knowledge about the exact molecular nature of this protein, however, remains fragmented. This review provides an overview of the structural research regarding CA125, and presents an orthogonal verification method to confirm the identity of this molecule. The need for independent identification of CA125 is exemplified by several reports where mutually exclusive data concerning the existence of isoforms and the glycan moieties is presented. Mass spectrometry can overcome the pitfalls of a single detection/identification method such as antibody probing. Independent verification of CA125 identity in characterization studies will help establish a refined model of its molecular structure that will promote the development of new approaches for diagnosis, prognosis and therapy of ovarian cancer.     

A Comparative Analysis of Genetic and Epigenetic Events of Breast and Ovarian Cancer Related to Tumorigenesis

Breast cancer persists as the most common cause of cancer death in women worldwide. Ovarian cancer is also a significant source of morbidity and mortality, as the fifth leading cause of cancer death among women. This reflects the continued need for further understanding and innovation in cancer treatment. Though breast and ovarian cancer usually present as distinct clinical entities, the recent explosion of large-scale -omics research has uncovered many overlaps, particularly with respect to genetic and epigenetic alterations. We compared genetic, microenvironmental, stromal, and epigenetic changes common between breast and ovarian cancer cells, as well as the clinical relevance of these changes. Some of the most striking commonalities include genetic alterations of BRCA1 and 2, TP53, RB1, NF1, FAT3, MYC, PTEN, and PIK3CA; down regulation of miRNAs 9, 100, 125a, 125b, and 214; and epigenetic alterations such as H3K27me3, H3K9me2, H3K9me3, H4K20me3, and H3K4me. These parallels suggest shared features of pathogenesis. Furthermore, preliminary evidence suggests a shared epigenetic mechanism of oncogenesis. These similarities, warrant further investigation in order to ultimately inform development of more effective chemotherapeutics, as well as strategies to circumvent drug resistance.     

新型癌抗原125化学发光免疫试剂盒的制备

目的制备新型癌抗原125(CA125)化学发光免疫试剂盒,并进行验证。方法用1株针对CA125蛋白特异性位点的单克隆抗体作为包被抗体,1株针对CA125分子糖链的碱性磷酸酶(ALP)标记单抗和另1株针对蛋白特异性位点的ALP标记单抗分别作为酶标抗体,采用双抗体夹心法,金刚烷增敏化学发光体系作为酶底物制备试剂盒,检测CA125,并对试剂盒进行验证。结果新型CA125试剂盒检测灵敏度达0.1U/ml,测定范围在0.1~430U/ml之间,与传统CA125试剂盒一致;与CA199、CA153和CA50糖蛋白无交叉反应;精密性和准确性良好。与传统CA125试剂盒比较,检测结果有显著性差异,特别是在卵巢癌标本检测方面,糖链单抗酶标抗体制备的试剂盒与普通抗体酶标抗体制备的试剂盒检测结果的比值与其他标本两者的比值之间差异具有统计学意义。结论已制备了新型CA125化学发光免疫试剂盒,其与传统试剂盒检测标本结果的比值更适用于对卵巢癌进行特异性诊断。     

Nipple Discharge of CA15-3, CA125, CEA and TSGF as a New Biomarker Panel for Breast Cancer

Breast cancer is the second leading cause of cancer death in women. Serum biomarkers such as cancer antigen 15-3 (CA15-3), cancer antigen 125 (CA125), and carcinoembryonic antigen (CEA) can be used as diagnostic and prognostic factors and can also provide valuable information during follow-up. However, serum protein biomarkers show limited diagnostic sensitivity and specificity in stand-alone assays because their levels reflect tumor burden. To validate whether biomarkers in nipple discharge may serve as novel biomarkers for breast cancer, we composed a panel of potential cancer biomarkers, including CA15-3, CA125, CEA, and malignant tumor-specific growth factor (TSGF), and evaluated their expression in both serum and nipple discharge in order to explore the expression and significance of estrogen receptor (ER), progestrone receptor (PR), epidermal growth factor receptor type 2 (HER2/neu), CA15-3, CA125, CEA, and TSGF expression for their combined predictive value for breast cancer and in judging the prognosis of breast cancer. Univariate analysis revealed that combined detection of CA15-3, CA125, CEA, and TSGF in nipple discharge served as novel biomarkers for the diagnosis and prognosis of breast cancer, but in the multivariate analyses the adverse effects of the four biomarkers combination in nipple discharge positivity on overall survival were lost. Multivariate analysis revealed that the positivity of the combined detection of the four biomarkers in both nipple discharge and serum was significantly higher than that of other detection methods. Thus, the combined detection of these four biomarkers both in serum and nipple discharge was retained as an independent prognostic variable in breast cancer patients. Our results indicate that CA15-3, CA125, CEA, and TSGF in nipple discharge can serve as novel biomarkers in the diagnosis and prognosis of breast cancer.     

Analysis of Composite Processing Using Magnetic Resonance Imaging

Nuclear magnetic resonance imaging utilizes FT-NMR in conjunction with linearly applied gradient fields to resolve spatially the spins in a protonated solution. Magnetic resonance imaging (MRI) has been used extensively in the medical field. More recently, MRI has been used to image the distribution of liquids in materials.^1-4 Protons are the most extensively used nuclei based on their sensitivity and abundance.     

A Brief History and Future Prospects of CEST MRI in Clinical Non-Brain Tumor Imaging

Chemical exchange saturation transfer (CEST) MRI is a promising molecular imaging tool which allows the specific detection of metabolites that contain exchangeable amide, amine, and hydroxyl protons. Decades of development have progressed CEST imaging from an initial concept to a clinical imaging tool that is used to assess tumor metabolism. The first translation efforts involved brain imaging, but this has now progressed to imaging other body tissues. In this review, we summarize studies using CEST MRI to image a range of tumor types, including breast cancer, pelvic tumors, digestive tumors, and lung cancer. Approximately two thirds of the published studies involved breast or pelvic tumors which are sites that are less affected by body motion. Most studies conclude that CEST shows good potential for the differentiation of malignant from benign lesions with a number of reports now extending to compare different histological classifications along with the effects of anti-cancer treatments. Despite CEST being a unique ‘label-free’ approach with a higher sensitivity than MR spectroscopy, there are still some obstacles for implementing its clinical use. Future research is now focused on overcoming these challenges. Vigorous ongoing development and further clinical trials are expected to see CEST technology become more widely implemented as a mainstream imaging technology.     

Functional Analysis of Familial Asp67Glu and Thr1051Ser BRCA1 Mutations in Breast/Ovarian Carcinogenesis

Estrogen is believed to be pre-initiator in the risk of breast cancer. The BRCA1 is a tumor suppressor gene associated with breast and ovarian cancer risk. This report describes functional analysis of two BRCA1 missense mutations (Asp67Glu and Thr1051Ser) observed in the familial breast/ovarian cancer patients in Thailand. Levels of luciferase activity of the two mutations were relatively lower than in the wild-type BRCA1. It is indicated that mutants may fail to promote the estrogen receptor dependent functions. It is presumed that estrogen and insulin/IGF-1 regulate c-Myc and cyclin D1 during breast cancer cell proliferation. It is also likely to affect ubiquitination mechanism. Since three affected cancer families carry the Asp67Glu mutation, it is believed that this type of mutation could have some effect on breast/ovarian cancer progression.     

Towards a New,Endophenotype-Based Strategy for Pathogenicity Prediction in BRCA1 and BRCA2: In Silico Modeling of the Outcome of HDR/SGE Assays for Missense Variants

The present limitations in the pathogenicity prediction of BRCA1 and BRCA2 (BRCA1/2) missense variants constitute an important problem with negative consequences for the diagnosis of hereditary breast and ovarian cancer. However, it has been proposed that the use of endophenotype predictions, i.e., computational estimates of the outcomes of functional assays, can be a good option to address this bottleneck. The application of this idea to the BRCA1/2 variants in the CAGI 5-ENIGMA international challenge has shown promising results. Here, we developed this approach, exploring the predictive performances of the regression models applied to the BRCA1/2 variants for which the values of the homology-directed DNA repair and saturation genome editing assays are available. Our results first showed that we can generate endophenotype estimates using a few molecular-level properties. Second, we show that the accuracy of these estimates is enough to obtain pathogenicity predictions comparable to those of many standard tools. Third, endophenotype-based predictions are complementary to, but do not outperform, those of a Random Forest model trained using variant pathogenicity annotations instead of endophenotype values. In summary, our results confirmed the usefulness of the endophenotype approach for the pathogenicity prediction of the BRCA1/2 missense variants, suggesting different options for future improvements.     

Magnetic Resonance Imaging of Tumor-Infiltrating Lymphocytes by Anti-CD3-Conjugated Iron Oxide Nanoparticles

Immune checkpoint blockade, considered a revolutionary approach in cancer treatment, is only effective in patients with high tumor-infiltrating lymphocytes (TILs). This work aimed to investigate the feasibility of targeted contrast agent (CA) based on dextran-coated superparamagnetic iron oxide nanoparticles (SPIONs-DEX) for TILs detection by magnetic resonance imaging (MRI) studies. To do so, we synthesized an MRI CA by conjugating SPIONs-DEX to an anti-CD3 monoclonal antibody via cyanogen bromide as a cross-linker. In vitro assessments demonstrated the higher labeling efficiency of the developed CA to CD3+ lymphocytes compared to SPIONs-DEX. In vivo MRI of a xenograft model of CD3+ lymphocytes revealed the significant signal loss after the intravenous injection of the bioconjugate by ∼34 % and 21 % in T₂*-weighted and T₂-weighted images, respectively. The histopathological evaluation of xenograft tumors confirmed the labeling of lymphocytes by the targeted CA. This approach could open up a new horizon in the non-invasive assessment of TILs to identify patients eligible for immunotherapy.     

A Case Report of Germline Compound Heterozygous Mutations in the BRCA1 Gene of an Ovarian and Breast Cancer Patient

The germline carrier of the BRCA1 pathogenic mutation has been well proven to confer an increased risk of breast and ovarian cancer. Despite BRCA1 biallelic pathogenic mutations being extremely rare, they have been reported to be embryonically lethal or to cause Fanconi anemia (FA). Here we describe a patient who was a 48-year-old female identified with biallelic pathogenic mutations of the BRCA1 gene, with no or very subtle FA-features. She was diagnosed with ovarian cancer and breast cancer at the ages of 43 and 44 and had a strong family history of breast and gynecological cancers.     

Electrochemical immunoassay for CA125 based on cellulose acetate stabilized antigen/colloidal gold nanoparticles membrane

A novel separation-free electrochemical immunosensor for carcinoma antigen-125 (CA125) was proposed based on the immobilization of CA125 antigen on colloidal gold nanoparticles that was stabilized with cellulose acetate membrane on a glassy carbon electrode. A competitive immunoassay format was employed to detect CA125 antigen with horseradish peroxidase (HRP) labeled CA125 antibody as tracer, o-phenylenediamine and hydrogen peroxide as enzyme substrates. After the immunosensor was incubated with a mixture of HRP labeled CA125 antibody and CA125 sample at 35 °C for 50 min, the amperometric response decreased with an increasing CA125 concentration in the sample solution. The decreased percentage of the electrocatalytic current was proportional to CA125 concentration ranging from 0 to 30 U ml⁻¹ with a detection limit of 1.73 U ml⁻¹ (S/N = 3). The proposed immunosensor showed good stability, acceptable accuracy, and would be applicable to clinical immunoassay of CA125.     

PARP Inhibitors Display Differential Efficacy in Models of BRCA Mutant High-Grade Serous Ovarian Cancer

Several poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitors are now in clinical use for tumours with defects in BReast CAncer genes BRCA1 or BRCA2 that result in deficient homologous recombination repair (HRR). Use of olaparib, niraparib or rucaparib for the treatment of high-grade serous ovarian cancer, including in the maintenance setting, has extended both progression free and overall survival for women with this malignancy. While different PARP inhibitors (PARPis) are mechanistically similar, differences are apparent in their chemical structures, toxicity profiles, PARP trapping abilities and polypharmacological landscapes. We have treated ovarian cancer cell line models of known BRCA status, including the paired cell lines PEO1 and PEO4, and UWB1.289 and UWB1.289+BRCA1, with five PARPis (olaparib, niraparib, rucaparib, talazoparib and veliparib) and observed differences between PARPis in both cell viability and cell survival. A cell line model of acquired resistance to veliparib showed increased resistance to the other four PARPis tested, suggesting that acquired resistance to one PARPi may not be able to be rescued by another. Lastly, as a proof of principle, HRR proficient ovarian cancer cells were sensitised to PARPis by depletion of BRCA1. In the future, guidelines will need to emerge to assist clinicians in matching specific PARPis to specific patients and tumours.     

A Bead-Based Multiplexed Immunoassay to Evaluate Breast Cancer Biomarkers for Early Detection in Pre-Diagnostic Serum

This study investigates whether a set of ten potential breast cancer serum biomarkers and cancer antigens (osteopontin (OPN), haptoglobin, cancer antigen 15-3 (CA15-3), carcinoembryonic antigen (CEA), cancer antigen 125 (CA-125), prolactin, cancer antigen 19-9 (CA19-9), α-fetoprotein (AFP), leptin and migration inhibitory factor (MIF)) can predict early stage breast cancer in samples collected before clinical diagnosis (phase III samples). We performed a nested case-control study within the Prospect-EPIC (European Prospective Investigation into Cancer and nutrition) cohort. We examined to what extent the biomarker panel could discriminate between 68 women diagnosed with breast cancer up to three years after enrollment and 68 matched healthy controls (all 56–64 years at baseline). Using a quantitative bead-based multiplexed assay, we determined protein concentrations in serum samples collected at enrollment. Principal Component Analysis (PCA) and Random Forest (RF) analysis revealed that on the basis of all ten proteins, early cases could not be separated from controls. When we combined serum protein concentrations and subject characteristics related to breast cancer risk in the RF analysis, this did not result in classification accuracy scores that could correctly classify the samples (sensitivity: 50%, specificity: 50%). Our findings indicate that this panel of selected tumor markers cannot be used for diagnosis of early breast cancer.     

Recent Advances in Bifunctional Paramagnetic Chelates for MRI

Magnetic resonance imaging (MRI) is a non-invasive diagnostic modality routinely employed in modern clinical medicine to collect images of the internal organs of the human body. Contrast agents (CA) are usually administered to patients to improve the sensitivity of this technique and nowadays these agents are gadolinium complexes. Bifunctional chelating agents (BFCAs) are dual molecules containing a multidentate ligand, able to strongly coordinate a metal ion, and a reactive moiety for conjugation purposes. With BFCAs an easy labelling of biomolecules or vectors with paramagnetic ions is possible, allowing the in vivo MRI visualization of diseases at cellular or molecular level.     

Breast Cancer Predisposition Genes and Synthetic Lethality

BRCA1 and BRCA2 are tumor suppressor genes with pivotal roles in the development of breast and ovarian cancers. These genes are essential for DNA double-strand break repair via homologous recombination (HR), which is a virtually error-free DNA repair mechanism. Following BRCA1 or BRCA2 mutations, HR is compromised, forcing cells to adopt alternative error-prone repair pathways that often result in tumorigenesis. Synthetic lethality refers to cell death caused by simultaneous perturbations of two genes while change of any one of them alone is nonlethal. Therefore, synthetic lethality can be instrumental in identifying new therapeutic targets for BRCA1/2 mutations. PARP is an established synthetic lethal partner of the BRCA genes. Its role is imperative in the single-strand break DNA repair system. Recently, Olaparib (a PARP inhibitor) was approved for treatment of BRCA1/2 breast and ovarian cancer as the first successful synthetic lethality-based therapy, showing considerable success in the development of effective targeted cancer therapeutics. Nevertheless, the possibility of drug resistance to targeted cancer therapy based on synthetic lethality necessitates the development of additional therapeutic options. This literature review addresses cancer predisposition genes, including BRCA1, BRCA2, and PALB2, synthetic lethality in the context of DNA repair machinery, as well as available treatment options.     

MUC16/CA125 in the Context of Modular Proteins with an Annotated Role in Adhesion-Related Processes: In Silico Analysis

Mucin 16 (MUC16) is a type I transmembrane protein, the extracellular portion of which is shed after proteolytic degradation and is denoted as CA125 antigen, a well known tumor marker for ovarian cancer. Regarding its polypeptide and glycan structures, as yet there is no detailed insight into their heterogeneity and ligand properties, which may greatly influence its function and biomarker potential. This study was aimed at obtaining further insight into the biological capacity of MUC16/CA125, using in silico analysis of corresponding mucin sequences, including similarity searches as well as GO (gene ontology)-based function prediction. The results obtained pointed to the similarities within extracellular serine/threonine rich regions of MUC16 to sequences of proteins expressed in evolutionary distant taxa, all having in common an annotated role in adhesion-related processes. Specifically, a homology to conserved domains from the family of herpesvirus major outer envelope protein (BLLF1) was found. In addition, the possible involvement of MUC16/CA125 in carbohydrate-binding interactions or cellular transport of protein/ion was suggested.     

Biomedical Applications of Fluorescent and Magnetic Resonance Imaging Dual-Modality Probes

Molecular imaging plays a critical role in biomedical research. The combination of different modalities can generate complementary information and provide synergistic advantages over single modality alone. Noninvasive and nonradioactive fluorescent imaging (FI)/magnetic resonance imaging (MRI) dualmodality probes fuse the high sensitivity of FI and the high temporal and spatial resolution and deep-tissue penetration of MRI, and their increasing applications have been reported in biomedical research and clinical practices, including cell labeling, enzyme activity measurement, tumor diagnosis and therapy, and anatomical localization and real-time assessment during surgery.     

Tumor-Specific miRNA Signatures in Combination with CA19-9 for Liquid Biopsy-Based Detection of PDAC

Pancreatic ductal adenocarcinoma (PDAC) is considered one of the most aggressive malignancies and has high mortality and poor survival rates. Therefore, there is an urgent need to discover non-invasive biomarkers for early detection before PDAC reaches the incurable stage. We hypothesized that liquid biopsy of PDAC-derived extracellular vesicles (PDEs) containing abundant microRNAs (miRNAs) could be used for early diagnosis of PDAC because they can be selectively enriched and because they are biologically stable. We isolated PDEs by immunocapture using magnetic beads, and we identified 13 miRNA candidates in 20 pancreatic cancer patients and 20 normal controls. We found that expression of five miRNAs, including miR-10b, miR-16, miR-155, miR-429, and miR-1290, was markedly higher in PDEs. Furthermore, the miRNA signatures along with serum carbohydrate antigen 19-9 (CA19-9) were optimized by logistic regression, and the miRNA signature and CA19-9 combination markers (CMs) were effective at differentiating PDAC patients from normal controls. As a result, the CMs represented a high sensitivity (AUC, 0.964; sensitivity, 100%; specificity, 80%) and a high specificity (AUC, 0.962; sensitivity, 85.71%; specificity, 100%). These findings suggest that five miRNAs expressed in PDEs and CA19-9 are valuable biomarkers for screening and diagnosis of pancreatic cancer by liquid biopsy.