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Background: Environmental pollution has emerged as a significant determinant in ovarian cancer prognosis. However, limited evidence exists regarding the correlations between heavy metals and ovarian cancer prognosis., Objective: To elucidate the relationship between urinary heavy metals and their mixtures with overall survival (OS) of advanced high-grade serous ovarian cancer (HGSOC)., Methods: Within the Ovarian Cancer Follow-Up Study, we conducted a nested case-control study. A sum of 159 deceased patients and an equal number of alive patients were included, matched by sample date, body mass index, and age at diagnosis. Urinary concentrations of five heavy metals were quantified: arsenic (As), cadmium (Cd), chromium (Cr), mercury (Hg), and lead (Pb). Conditional logistic regression models were employed to calculate odds ratios (ORs) and their 95 % confidence intervals (CIs). To elucidate joint effects, we utilized quantile g-computation and Bayesian kernel machine regression models., Results: For the multivariable adjusted conditional logistic regression model, significant associations were found between high urinary levels of As (OR=1.99, 95 %CI: 1.05-3.79), Cd (OR=2.56, 95 %CI: 1.29-5.05), Hg (OR=2.24, 95 %CI: 1.09-4.62), and Pb (OR=3.80, 95 %CI: 1.75-8.27) and worse OS of HGSOC, comparing the highest tertile to the lowest. Analysis of joint effects showed that elevated concentrations of heavy metal mixtures were related to poor OS of HGSOC. Pb exhibited the highest contribution to the overall association within the metal mixtures., Conclusions: High urinary heavy metal concentrations were linked to worse OS of HGSOC. Future research is necessary to validate our findings., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Ovarian cancer is the 8th most common malignancy in women and the deadliest gynecological cancer. Due to the non-specific symptoms and the lack of effective diagnostic methods, late diagnosis remains the main barrier for improving the poor prognosis. Human epididymis protein 4 (HE4) is a protein overexpressed in ovarian cancer, but not in healthy individuals or benign conditions. The aim of this review article is to evaluate the laboratory aspect and potential clinical application of urine HE4. The methodology is presented, together with discussion on preanalytical, analytical and postanalytical phase of HE4 detection using urine. Moreover, we present the diagnostic role of urine HE4 in differential diagnosis, chemotherapy response, detection of recurrence and detection of low-malignant potential tumors. It has been found that urine HE4 presents as a promising, non-invasive tumor marker for detection and monitoring of ovarian cancer. However, standardization of the HE4 detection process is needed prior to implementation in clinical diagnostics., Competing Interests: Potential conflict of interest None declared., (Croatian Society of Medical Biochemistry and Laboratory Medicine.)

High-grade serous ovarian cancer (HGSOC) is one of the most common histologic types of ovarian cancer. The purpose of this study was to identify potential prognostic biomarkers in urine specimens from patients with HGSOC. First, 56 urine samples with information on relapse-free survival (RFS) months were collected and classified into good prognosis (RFS ≥ 12 months) and poor prognosis (RFS < 12 months) groups. Next, data-independent acquisition (DIA)-based mass spectrometry (MS) analysis was combined with MSFragger-DIA workflow to identify potential prognostic biomarkers in a discovery set (n = 31). With the aid of parallel reaction monitoring (PRM) analysis, four candidate biomarkers (ANXA1, G6PI, SPB3, and SPRR3) were finally validated in both the discovery set and an independent validation set (n = 25). Subsequent RFS and Cox regression analyses confirmed the utility of these candidate biomarkers as independent prognostic factors affecting RFS in patients with HGSOC. Regression models were constructed to predict the 12-month RFS rate, with area under the receiver operating characteristic curve (AUC) values ranging from 0.847 to 0.905. Overall, candidate prognostic biomarkers were identified in urine specimens from patients with HGSOC and prediction models for the 12-month RFS rate constructed. SIGNIFICANCE: OC is one of the leading causes of death due to gynecological malignancies. HGSOC constitutes one of the most common histologic types of OC with aggressive characteristics, accounting for the majority of advanced cases. In cases where patients with advanced HGSOC potentially face high risk of unfavorable prognosis or disease advancement within a 12-month period, intensive medical monitoring is necessary. In the era of precision cancer medicine, accurate prediction of prognosis or 12-month RFS rate is critical for distinguishing patient groups requiring heightened surveillance. Patients could significantly benefit from timely modifications to treatment regimens based on the outcomes of clinical monitoring. Urine is an ideal resource for disease surveillance purposes due to its easy accessibility. Furthermore, molecules excreted in urine are less complex and more stable than those in other liquid samples. In the current study, we identified candidate prognostic biomarkers in urine specimens from patients with HGSOC and constructed prediction models for the 12-month RFS rate., Competing Interests: Declaration of competing interest There are no potential conflicts of interest to declare. The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Ovarian cancer remains the most lethal gynaecological malignancy, as its timely detection at early stages remains elusive. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy of biofluids has been previously applied in pilot studies for ovarian cancer diagnosis, with promising results. Herein, these initial findings were further investigated by application of ATR-FTIR spectroscopy in a large patient cohort. Spectra were obtained by measurements of blood plasma and serum, as well as urine, from 116 patients with ovarian cancer and 307 patients with benign gynaecological conditions. A preliminary chemometric analysis revealed significant spectral differences in ovarian cancer patients without previous chemotherapy (n = 71) and those who had received neo-adjuvant chemotherapy-NACT (n = 45), so these groups were compared separately with benign controls. Classification algorithms with blind predictive model validation demonstrated that serum was the best biofluid, achieving 76% sensitivity and 98% specificity for ovarian cancer detection, whereas urine exhibited poor performance. A drop in sensitivities for the NACT ovarian cancer group in plasma and serum indicates the potential of ATR-FTIR spectroscopy to identify chemotherapy-related spectral changes. Comparisons of regression coefficient plots for identification of biomarkers suggest that glycoproteins (such as CA125) are the main classifiers for ovarian cancer detection and responsible for smaller differences in spectra between NACT patients and benign controls. This study confirms the capacity of biofluids' ATR-FTIR spectroscopy (mainly blood serum) to diagnose ovarian cancer with high accuracy and demonstrates its potential in monitoring response to chemotherapy, which is reported for the first time. ATR-FTIR spectroscopy of blood serum achieves good segregation of ovarian cancers from benign controls, with attenuation of differences following neo-adjuvant chemotherapy., (© 2021. The Author(s).)

Ovarian cancer is often diagnosed in an advanced stage and is associated with a high mortality rate. It is assumed that early detection of ovarian cancer could improve patient outcomes. Unfortunately, effective screening methods for early diagnosis of ovarian cancer are still lacking. Extracellular RNAs circulating in human biofluids can reliably be measured and are emerging as potential biomarkers in cancer. In this systematic review, we present 75 RNA biomarkers detectable in human biofluids that have been studied for early diagnosis of ovarian cancer. The majority of these markers are microRNAs identified using RT-qPCR or microarrays in blood-based fluids. A handful of studies used RNA-sequencing and explored alternative fluids, such as urine and ascites. Candidate RNA biomarkers that were more abundant in biofluids of ovarian cancer patients compared to controls in at least two independent studies include miR-21, the miR-200 family, miR-205, miR-10a and miR-346. Amongst the markers confirmed to be lower in at least two studies are miR-122, miR-193a, miR-223, miR-126 and miR-106b. While these biomarkers show promising diagnostic potential, further validation is required before implementation in routine clinical care. Challenges related to biomarker validation and reflections on future perspectives to accelerate progress in this field are discussed., Competing Interests: Declaration of Competing Interest The authors have no competing interests to declare., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective: Plasma, but also urine sample could represent a simple liquid biopsy for ovarian cancer biomarker detection. The miRNA-200 family has been shown to be dysregulated in ovarian cancer. The aim of this study was to isolate three members of miR-200 family from tumor tissue, plasma and urine of high-grade serous ovarian cancer patients in comparison with samples from patients with benign ovarian tumors. This is a methodological pilot study of a prospective ovarian cancer patient cohort investigating the potential of liquid biopsies and the role of miRNAs in ovarian cancer treatment., Results: MiR-200a, miR-200b and miR-200c were isolated from samples of nine ovarian cancer patients and seven patients with benign ovarian tumor. The most significant finding is that all three miRNAs were detectable in all sample types. Tumor tissue and plasma, but not urine analysis was able to discriminate malignant and benign samples. A correlation between the miRNA-200 expression in urine and plasma was observed in malignant samples only. Plasma and urine with respect to miRNA detection show potential according to this study, but larger studies are needed to clarify the usefulness of these liquid biopsies in ovarian cancer., Trial Registration: ClinicalTrials.gov NCT02758652, May 2, 2016.

Background: Deregulated microRNAs (miRNAs) in breast and gynecological cancer might contribute to improve early detection of female malignancies., Objective: Specification of miRNA types in serum and urine as minimally-invasive biomarkers for breast (BC), endometrial (EC) and ovarian cancer (OC)., Methods: In a discovery phase, serum and urine samples from 17 BC, five EC and five OC patients vs. ten healthy controls (CTRL) were analyzed with Agilent human miRNA microarray chip. Selected miRNA types were further investigated by RT-qPCR in serum (31 BC, 13 EC, 15 OC patients, 32 CTRL) and urine (25 BC, 10 EC, 10 OC patients, 30 CTRL) applying two-sample t-tests., Results: Several miRNA biomarker candidates exhibited diagnostic features due to distinctive expression levels (serum: 26; urine: 22). Among these, miR-518b, -4719 and -6757-3p were found specifically deregulated in BC serum. Four, non-entity-specific, novel biomarker candidates with unknown functional roles were identified in urine (miR-3973; -4426; -5089-5p and -6841). RT-qPCR identified miR-484/-23a (all p⩽ 0.001) in serum as potential diagnostic markers for EC and OC while miR-23a may also serve as an endogenous control in BC diagnosis., Conclusions: Promising miRNAs as liquid biopsy-based tools in the detection of BC, EC and OC qualified for external validation in larger cohorts.

This study was designed to analyze urinary proteins associated with ovarian cancer (OC) and investigate the potential urinary biomarker panel to predict malignancy in women with pelvic masses. We analyzed 23 biomarkers in urine samples obtained from 295 patients with pelvic masses scheduled for surgery. The concentration of urinary biomarkers was quantitatively assessed by the xMAP bead-based multiplexed immunoassay. To identify the performance of each biomarker in predicting cancer over benign tumors, we used a repeated leave-group-out cross-validation strategy. The prediction models using multimarkers were evaluated to develop a urinary ovarian cancer panel. After the exclusion of 12 borderline tumors, the urinary concentration of 17 biomarkers exhibited significant differences between 158 OCs and 125 benign tumors. Human epididymis protein 4 (HE4), vascular cell adhesion molecule (VCAM), and transthyretin (TTR) were the top three biomarkers representing a higher concentration in OC. HE4 demonstrated the highest performance in all samples withOC(mean area under the receiver operating characteristic curve (AUC) 0.822, 95% CI: 0.772-0.869), whereas TTR showed the highest efficacy in early-stage OC (AUC 0.789, 95% CI: 0.714-0.856). Overall, HE4 was the most informative biomarker, followed by creatinine, carcinoembryonic antigen (CEA), neural cell adhesion molecule (NCAM), and TTR using the least absolute shrinkage and selection operator (LASSO) regression models. A multimarker panel consisting of HE4, creatinine, CEA, and TTR presented the best performance with 93.7% sensitivity (SN) at 70.6% specificity (SP) to predict OC over the benign tumor. This panel performed well regardless of disease status and demonstrated an improved performance by including menopausal status. In conclusion, the urinary biomarker panel with HE4, creatinine, CEA, and TTR provided promising efficacy in predicting OC over benign tumors in women with pelvic masses. It was also a non-invasive and easily available diagnostic tool.

Introduction: Our primary objective was to test the hypothesis that human epididymal protein 4 (HE4) and risk of ovarian malignancy index outperform the CA 125 and risk of malignancy index tests in categorizing a pelvic mass into high or low risk of malignancy in a Swedish population. Furthermore, cut-off values needed to be defined for HE4 and ROMA in premenopausal and postmenopausal women prior to their introduction to clinical practice. A third objective was to investigate the correlation between HE4 levels in serum and urine., Material and Methods: Women with a pelvic mass scheduled for surgery were recruited from nine hospitals in south-east Sweden. Preoperative blood samples were taken for analyzing CA125 and HE4 as well as urine samples for analyzing HE4., Results: We enrolled a total of 901 women, of whom 784 were evaluable. In the premenopausal and postmenopausal groups, no significant differences were found for sensitivity, positive and negative predictive value, either for RMI vs ROMA or for CA125 vs HE4 using a fixed specificity of 75%. Cut-off values indicating malignancy were established for HE4 and ROMA in premenopausal and postmenopausal women. We found no correlation between HE4 concentration in serum and urine., Conclusions: We could not confirm that ROMA had diagnostic superiority over RMI in categorizing women with a pelvic mass into low-risk or high-risk groups for malignancy in a Swedish population. We have defined cut-off values for HE4 and ROMA. The lack of correlation between serum and urine HE4 obviates the introduction of urine HE4 analysis in clinical diagnostics., (© 2018 Nordic Federation of Societies of Obstetrics and Gynecology.)

Aim: We hypothesized that field asymmetric waveform ion mobility spectrometry (FAIMS) as a novel artificial olfactory technology could differentiate urine of women with malignant ovarian tumors from controls and women with benign tumors, based on previous findings on the ability of canine olfactory system to "smell" cancer., Patients and Methods: Preoperative urine samples from 51 women with ovarian tumors, both benign and malignant, and from 18 women with genital prolapse, as controls, were collected. The samples were analyzed by FAIMS device. Data analysis was processed by quadratic data analysis (QDA) and linear discriminant analysis (LDA), and cross-validated using 10-fold cross-validation., Results: Thirty-three women had malignant ovarian tumors, of which 18 were high-grade cancers. FAIMS distinguished controls from malignancies with the accuracy of 81.3% (sensitivity 91.2% and specificity 63.1%), and benign tumors from malignancies with the accuracy of 77.3% (sensitivity 91.5% and specificity 51.4%). Moreover, low grade tumors were also separated from high grade cancers and benign ovarian tumors with accuracies of 88.7% (sensitivity 87.8% and specificity 89.6%) and 83.9% (sensitivity 73.1% and specificity 92.9%), respectively., Conclusions: This proof of concept-study indicates that the FAIMS from urine has potential to discriminate malignant ovarian tumors from no tumor-bearing controls and benign tumors., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The current lack of an accurate, cost-effective and non-invasive test that would allow for screening and diagnosis of gynaecological carcinomas, such as endometrial and ovarian cancer, signals the necessity for alternative approaches. The potential of spectroscopic techniques in disease investigation and diagnosis has been previously demonstrated. Here, we used attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy to analyse urine samples from women with endometrial (n = 10) and ovarian cancer (n = 10), as well as from healthy individuals (n = 10). After applying multivariate analysis and classification algorithms, biomarkers of disease were pointed out and high levels of accuracy were achieved for both endometrial (95% sensitivity, 100% specificity; accuracy: 95%) and ovarian cancer (100% sensitivity, 96.3% specificity; accuracy 100%). The efficacy of this approach, in combination with the non-invasive method for urine collection, suggest a potential diagnostic tool for endometrial and ovarian cancers.

Purpose: For the vast majority of ovarian cancer patients, optimal surgical debulking remains a key prognostic factor associated with improved survival. A standardized, biomarker-based test, to preoperatively discriminate benign from malignant disease and inform appropriate patient triage, is highly desirable. However, no fit-for-purpose biomarkers have yet been identified., Experimental Design: We conducted a pilot study consisting of 40 patient urine samples (20 from each group), using label-free quantitative (LFQ) mass spectrometry, to identify potential biomarker candidates in urine from individual ovarian cancer patients. To validate these changes, we used parallel reaction monitoring (PRM) to investigate their abundance in an independent validation cohort (n = 20) of patient urine samples., Results: LFQ analyses identified 4394 proteins (17 027 peptides) in a discovery set of 20 urine samples. Twenty-three proteins were significantly elevated in the malignant patient group compared to patients with benign disease. Several proteins, including LYPD1, LYVE1, PTMA, and SCGB1A1 were confirmed to be enriched in the urine of ovarian cancer patients using PRM. We also identified the established ovarian cancer biomarkers WFDC2 (HE4) and mesothelin (MSLN), validating our approach., Conclusions and Clinical Relevance: This is the first application of a LFQ-PRM workflow to identify and validate ovarian cancer-specific biomarkers in patient urine samples., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Readily accessible samples such as urine or blood are seemingly ideal for differentiating and stratifying patients; however, it has proven a daunting task to identify reliable biomarkers in such samples. Noncoding RNA holds great promise as a source of biomarkers distinguishing physiologic wellbeing or illness. Current methods to isolate and characterize RNA molecules in urine are limited. In this proof of concept study, we present a method to extract and identify small noncoding RNAs in urine. Initially, quantitative reverse transcription PCR was applied to confirm the presence of microRNAs in total RNA extracted from urine. Once the presence of micro RNA in urine was confirmed, we developed a method to scale up RNA extraction to provide adequate amounts of RNA for next generation sequence analysis. The method described in this study is applicable to detecting a broad range of small noncoding RNAs in urine; thus, they have wide applicability for health and disease analyses., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objectives: Elevated concentrations of polyamines have been found in urine of patients with malignant tumors, including ovarian cancer. Previous research has suffered from poorly standardized detection methods. Our liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is capable of simultaneous standardized analysis of most known polyamines. Liquid chromatography-tandem mass spectrometry has not previously been used in the differential diagnostics of ovarian tumors in postmenopausal women., Materials and Methods: In this prospective study, postmenopausal women (n = 71) presenting with an adnexal mass and, as controls, women with genital prolapse or urinary incontinence scheduled for surgery (n = 22) were recruited in the study. For analysis of the polyamines, a morning urine sample was obtained before surgery. Preoperative serum CA125 concentrations were determined in the study group., Results: Twenty-three women with benign and 37 with malignant ovarian tumors were eligible. Of all analyzed polyamines, only urinary N,N-diacetylspermine showed statistically significant differences between all groups except controls versus benign tumors. N,N-diacetylspermine was elevated in malignant versus benign tumors (P < 0.001), in high-grade versus low malignant potential tumors (P < 0.001), in stage III to IV versus stage I to II cancers (P < 0.001), and even in early-stage cancer (stage I-II) versus benign tumors (P = 0.017). N,N-diacetylspermine had better sensitivity (86.5%) but lower specificity (65.2%) for distinguishing benign and malignant ovarian tumors than CA125 with a cut-off value of 35 kU/L (sensitivity, 75.7%; specificity, 69.6%)., Conclusions: Urinary N,N-diacetylspermine seems to be able to distinguish benign and malignant ovarian tumors as well as early and advanced stage, and low malignant potential and high-grade ovarian cancers from each other, respectively.

Data on early markers for acute kidney injury (AKI) after noncardiovascular surgery are still limited. This study aimed to determine the diagnostic value of plasma neutrophil-gelatinase-associated lipocalin (pNGAL) and intraoperative diuresis for AKI in patients undergoing major abdominal surgery treated within a goal-directed hemodynamic algorithm.This study is a post-hoc analysis of a randomized controlled pilot trial comparing intravenous solutions within a hemodynamic goal-directed algorithm based on the esophageal Doppler in patients undergoing epithelial ovarian cancer surgery. The diagnostic value of plasma NGAL obtained at ICU admission and intraoperative diuresis was determined with respect to patients already meeting AKI criteria 6 hours after surgery (AKI6h) and to all patients meeting AKI criteria at least once during the postoperative course (AKItotal). AKI was diagnosed by the definition of the Kidney Disease Improving Global Outcome (KDIGO) group creatinine criteria and was screened up to postoperative day 3. Receiver operating characteristic curves including a gray zone approach were performed.A total of 48 patients were analyzed. None of the patients had increased creatinine levels before surgery and 14 patients (29.2%) developed AKI after surgery. Plasma NGAL was predictive for AKI6h (AUCAKI6h 0.832 (95% confidence interval [CI], 0.629-0.976), P = .001) and AKItotal (AUCAKItotal 0.710 (CI 0.511-0.878), P = .023). The gray zones of pNGAL calculated for AKI6h and AKItotal were 210 to 245 and 207 to 274 ng mL, respectively. The lower cutoffs of the gray zone at 207 and 210 ng mL had a negative predictive value (NPV) (i.e., no AKI during the postoperative course) of 96.8% (CI 90-100) and 87.1% (CI 78-97), respectively. Intraoperative diuresis was also predictive for AKI6h (AUCAKI6h 0.742 (CI 0.581-0.871), P = .019) with a gray zone of 0.5 to 2.0 mL kg h. At the lower cutoff of the gray zone at 0.5 mL kg h, corresponding to the oliguric threshold, the NPV was 84.2% (78-92).This study indicates that pNGAL can be used as an early marker to rule out AKI occurring within 3 days after major abdominal surgery. Intraoperative diuresis can be used to rule out AKI occurring up to 6 hours after surgery., Trial Registration: ISRCTN 53154834.

Background: Granulosa cell tumor of the ovary is the most frequent sex cord stromal tumor and represents 2 to 5% of all primary ovarian cancers. Ovarian granulosa cell tumor is a malignant tumor with slow progression and in some cases this tumor is hormonally active. The recurrence of granulosa cell tumor often happens after 5 years., Case Presentation: We describe two cases of postmenopausal women with adult-type granulosa cell tumors of the ovary. Patient 1 is a 49-year-old European woman with a recurrent tumor; patient 2 is a 55-year-old European woman without recurrence of tumor. Urinary steroid profiles of patient 1 were monitored during a 5-year period starting from before an operation (13 samples). In patient 2, the urinary steroid profiles were monitored during a 3-year period starting from after an operation (six samples). The 24-hour urinary samples were examined and the urinary concentration of 20 androgen, progesterone, and corticoid metabolites was quantitatively determined by gas chromatography-mass spectrometry with selected ion-monitoring mode., Conclusions: Based on these cases a correlation could be observed between increased levels of the urinary steroids and the recurrence of ovarian granulosa cell tumor; therefore, we concluded that a urinary steroid profile could be a more effective method to follow-up such patients compared to the traditional serum hormones determinations supplemented with conventional tumor markers.

Breast cancer is the most common malignancy in women worldwide, and ovarian cancer is the most lethal gynecological malignancy. Women carrying a BRCA1/2 mutation have a very high lifetime risk of developing breast and ovarian cancer. The only effective risk-reducing strategy in BRCA-mutated women is a prophylactic surgery with bilateral mastectomy and bilateral salpingo-oophorectomy. However, many women are reluctant to undergo these prophylactic surgeries due to a consequent mutilated body perception, unfulfilled family planning, and precocious menopause. In these patients, an effective screening strategy is available only for breast cancer, but it only consists in close radiological exams with a significant burden for the health system and a significant distress to the patients. No biomarkers have been shown to effectively detect breast and ovarian cancer at an early stage. MicroRNAs (miRNAs) are key regulatory molecules operating in a post-transcriptional regulation of gene expression. Aberrant expression of miRNAs has been documented in several pathological conditions, including solid tumors, suggesting their involvement in tumorigenesis. miRNAs can be detected in blood and urine and could be used as biomarkers in solid tumors. Encouraging results are emerging in gynecological malignancy as well, and suggest a different pattern of expression of miRNAs in biological fluids of breast and ovarian cancer patients as compared to healthy control. Aim of this study is to highlight the role of the urinary miRNAs which are specifically associated with cancer and to investigate their role in early diagnosis and in determining the prognosis in breast and ovarian cancer.

Introduction: Serum epididymis protein 4 (HE4) level is a useful biomarker for the management of ovarian and endometrial cancer patients. Urine HE4-test, with its easier access than serum test, has emerged as a new method with promising application for the diagnosis of ovarian cancer. Areas covered: This review summarizes data regarding the detection and alteration of HE4 in urine samples collected from ovarian cancer patients and controls. The performance and limitation of the assay and potential direction of future study are also discussed. Expert commentary: Several studies have demonstrated an appreciable efficiency of urine HE4-test in the discrimination of ovarian cancer patients from general population. However, the data is based on small cohorts, and the performance of urine HE4-test need to be validated in larger groups. An algorithm incorporating other important factors may allow a quantitative assessment of cancer possibility. Future studies on the HE4 renal secretion and HE4 degradation dynamics in urine are also required for the establishment of standard protocols for the application of urine HE4-test in clinical settings.

Urine HE4 has been reported as the potential novel diagnostic biomarker for ovarian cancer in several studies, but their results were inconsistent. Therefore, we conducted a systematic analysis to evaluate the diagnostic value of urine HE4 in detecting ovarian cancer. A comprehensive electronic and manual search was conducted for relevant literatures through several databases up to May 5, 2016. The quality of the studies included in the systematic review was assessed using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool. All analyses were conducted using Meta-DiSc 1.4 and STATA 12.0 software. A total of seven publications were included in this study, and these studies included 413 ovarian cancer patients and 573 controls. The summary estimates were: sensitivity 0.76 (95% confidence interval [CI]: 0.72-0.80), specificity 0.92 (95% CI: 0.89-0.94), positive likelihood ratio 8.39 (95%CI: 4.81-14.63), negative likelihood ratio 0.23 (95% CI: 0.13-0.39), diagnostic odds ratio 37.90 (95% CI: 18.69-76.83), and area under the curve 0.93. According to our results, urine HE4 has greater diagnostic value in detecting ovarian cancer. In addition, considering the high heterogeneity, further research studies with more well-designed and large sample sizes are needed in the future.

Cell-free microRNAs (miRNAs) have become one of the novel promising diagnostic and prognostic biomarkers for various diseases recently. Blood serum and plasma along with urine are the most common sources of clinically well, almost noninvasively available samples containing various types of miRNAs. Here, we present a protocol for a small-scale study investigating expression of several candidate miRNAs. Small-scale experiments may be worth investigating in cases where no information is available on miRNAs expression in particular diseases, for validation of previously published miRNAs with promising diagnostic potential, particularly in situations where follow-up study is aimed at validating miRNAs coming from array or NGS experiments, or where funding for these large-scale experiments is not available.Using urine miRNAs expression as the novel diagnostic tools is challenging and currently this approach is still in its infancy. Therefore, various methods may result in different conclusions depending on clinical sample sets and differences among methods used for the miRNAs isolation and quantitation. In this protocol, we present the method evaluated in the study focused on cell-free urinary miRNAs in ovarian and endometrial cancers. We recommend using stabilization tubes for the urine collection, as this step may be necessary to stop activity of RNases. Further, routine real-time PCR methods are described. We demonstrate that assessment of urinary miRNAs expression may reveal as a feasible method to explore the potential for finding novel diagnostic and prognostic markers.

Background: Neopterin is produced by activated macrophages upon stimulation with interferon-γ (IFN-γ) and thus, elevated neopterin concentrations in patients indicate cellular inate immune response. Most studies in patients with malignant diseases found an association between higher neopterin concentrations and reduced survival and impaired prognosis. Nevertheless, neopterin is not a classical tumor marker since it is not produced by the cancer cells themselves., Patients and Methods: In a study conducted by the Austrian Gynecologic Oncology Group (AGO) in 114 patients with ovarian cystadenomas and 223 patients with invasive ovarian cancer, patients' urinary neopterin was determined before and after primary therapy. The relevance of neopterin in long-term median follow-up was assessed., Results: Elevated levels (cut-off 250 µmol/mol creatinine) were found less frequently in women with benign ovarian cystadenomas (24%) than in patients with malignant disease (58%). After 10 years, only 57% of ovarian cancer patients with elevated urinary neopterin levels survived without disease progression following primary therapy when compared with 86% of women with normal levels (P < 0.001). Along with residual tumor, FIGO stage, age and histological type, neopterin was significantly associated with overall survival (OS) and progression-free survival (PFS). The median PFS was 52 and 12 months and the median OS was 81 and 24 months for patients with normal and elevated neopterin, respectively, P < 0.001. In a multivariate Cox regression analysis, only residual tumor, neopterin and age were independently associated with OS, while only residual tumor was predictive for PFS. Thirty patients with early-stage invasive ovarian cancer (FIGO I and II) were analyzed separately. Of 3 patients with elevated neopterin, 2 died of disease in contrast to 2 out of 27 patients with normal neopterin (P = 0.004)., Conclusion: In ovarian cancer, the negative impact of elevated urinary neopterin levels indicates a detrimental effect of cancer-associated inflammatory reaction., (© The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Purpose: Endometrial (ECa), ovarian (OCa) and cervical (CCa) cancers are among 10 of the most common cancers affecting women worldwide. Cancers are known to cause some proteins to be differentially glycosylated or aberrantly excreted in the urine, which can be used as biomarkers. Since ECa, OCa and CCa are difficult to diagnose at the early stage, the aim of the present study was to identify a panel of new biomarkers for early detection of the cancers using surface-enhanced laser desorption/ionization-time-of-flight (SELDI-TOF) technology. Identification of early biomarkers that are specific and efficient can increase the survival rate of the patients., Experimental Design: Digested urinary proteins from patients with ECa, OCa and CCa were incubated on the champedak mannose-binding (CMB) lectin-immobilized PS10 chip. The lectin-captured glycopeptides were detected with SELDI-TOF mass spectrometry and followed by biomarker wizard analysis., Results: Peaks m/z 1201 and 1449 were detected as potential group discriminators. The peak m/z 1201 could distinguish OCa from CCa and ECa and its sensitivity and specificity were 100%. For m/z 1449, it was able to differentiate ECa from the other two types of cancer., Conclusions: The findings of this study suggest urinary glycopeptides m/z 1201 and 1449 may serve as potential biomarkers for the early detection of ECa, OCa and CCa, although this requires further extensive validation on clinically representative populations.

MicroRNAs (miRNAs) are key regulatory molecules implicated in fundamental cell processes. Recent investigations have been focused to investigate their diagnostic potential also in various body fluids. Plasma and serum are widely used for these purposes. Urinary miRNAs, as the easily available type of sample, have been explored particularly in urological diseases recently. However, we have shown previously that differential expression of urinary cell-free miRNAs may be observed also in gynaecological cancers, such as ovarian and endometrial cancers. In the present article, we focus on the differences in particular urine cell-free miRNA abundance among different samples including particularly ovarian and endometrial cancers and rare gynaecological diagnoses involved in the study. Using raw abundance miRNA expression data, we confirmed significant up-regulation of miR-92a in ovarian cancer, and significant down-regulation of miR-106b in endometrial cancers. As miR-21 appeared up-regulated in the endometrial cancer similarly as in the verification process, where also miR-106b resulted in significant down-regulation in ovarian cancer, these miRNAs may be good candidates for further evaluation as novel diagnostics. To find out why supernatant but not exosomal urine miRNAs fraction resulted in significant results in regards to de-regulation of expression, we performed a comparison of the same urine samples isolated by these two manners. We show that diagnostic potential of cell-free urinary miRNAs may depend on the urine fraction used for the isolation. While particular urinary miRNAs may be enriched, other may reveal unchanged or diminished expression in the exosomal fraction in comparison with supernatant fraction, giving differences also between cancer and control samples. More research will be needed to further explore which kind of cell-free samples would give better results for diagnostic purposes in various diagnoses using urinary samples and investigating cell-free miRNAs expression. Meanwhile, different urine fractions should be explored for their miRNA expression to establish novel diagnostic urinary miRNA markers.

Among gynaecological cancers, epithelial ovarian cancers are the most deadly cancers while endometrial cancers are the most common diseases. Efforts to establish relevant novel diagnostic, screening and prognostic markers are aimed to help reduce the high level of mortality, chemoresistance and recurrence, particularly in ovarian cancer. MicroRNAs, the class of post-transcriptional regulators, have emerged as the promising diagnostic and prognostic markers associated with various diseased states recently. Urine has been shown as the source of microRNAs several years ago; however, there has been lack of information on urine microRNA expression in ovarian and endometrial cancers till now. In this pilot study, we examined the expression of candidate cell-free urine microRNAs in ovarian cancer and endometrial cancer patients using quantitative real-time PCR. We compared the expression between pre- and post-surgery ovarian cancer samples, and between patients with ovarian and endometrial cancers and healthy controls, within three types of experiments. These experiments evaluated three different isolation methods of urine RNA, representing two supernatant and one exosome fractions of extracellular microRNA. In ovarian cancer, we found miR-92a significantly up-regulated, and miR-106b significantly down-regulated in comparison with control samples. In endometrial cancer, only miR-106b was found down-regulated significantly compared to control samples. Using exosome RNA, no significant de-regulations in microRNAs expression could be found in either of the cancers investigated. We propose that more research should now focus on confirming the diagnostic potential of urine microRNAs in gynaecological cancers using more clinical samples and large-scale expression profiling methods.

Objectives: To measure HE4 levels in urine from normal donors, patients with LMP tumors and ovarian cancer patients and to correlate levels with clinical factors in ovarian cancer patients., Methods: Archived samples from controls, patients with LMP tumors and ovarian cancer were tested using commercial assays, including serially collected serum and urine samples from women treated for stage III/IV serous ovarian cancer., Results: Five of 6 patients with stage I/II and 26 of 36 stage III/IV serous ovarian cancer patients had HE4-positive urines, similar to serum samples (4 of 5 stage I/II and 26 of 34 stage III/IV) when tested at the same level of specificity (95%). Urine HE4 was more sensitive in LMP tumors: 9 of 32 urines (28%) HE4-positive versus 3 of 68 sera (4.4%, p=0.002). Mean levels of serum CA125 and HE4 decreased comparably in patients during initial treatment regardless of their primary platinum response, but mean urine HE4 levels decreased only 7% in primary platinum resistant patients while decreasing 68% in those who were sensitive. By 7months after diagnosis, urine HE4 levels were higher in primary platinum resistant patients compared to those who proved to be sensitive (p=0.051) and persisted 12months after diagnosis (p=0.014). HE4 values in urine also became positive in advance of clinical recurrence in several women while serum HE4 and serum CA-125 remained normal., Conclusions: Measuring HE4 in urine complements serum assays for the detection of ovarian cancer and may allow identification of patients at high risk for primary platinum resistance., (Copyright © 2015 Elsevier Inc. All rights reserved.)

MicroRNAs (miRNAs) can serve as biomarkers in human cancer. To determine the clinical value of urinary miRNAs for ovarian serous adenocarcinoma, we collected urine samples from 39 ovarian serous adenocarcinoma patients, 26 patients with benign gynecological disease and 30 healthy controls. The miRNA microarray data showed that only miR-30a-5p was upregulated and 37 miRNAs were downregulated in the urine samples of ovarian serous adenocarcinoma patients, when compared to healthy controls, which was confirmed after conducting quantitative PCR. The upregulation of urinary miR-30a-5p was closely associated with early stage of ovarian serous adenocarcinoma as well as lymphatic metastasis. Receiver operator characteristic (ROC) analysis demonstrated the potential use of urinary miR-30a-5p as a diagnostic marker for ovarian serous adenocarcinoma. Furthermore, a lower urine level of miR-30a-5p was found in 20 gastric cancer and 20 colon carcinoma patients when compared to ovarian serous adenocarcinoma, suggesting that the upregulation of urinary miR-30a-5p may be specific for ovarian serous adenocarcinoma. miR-30a-5p was also upregulated in ovarian serous adenocarcinoma tissues and cell lines, while urinary miR-30a-5p from ovarian cancer patients was notably reduced following the surgical removal of ovarian serous adenocarcinoma, suggesting that urinary miR-30a-5p was derived from the ovarian serous adenocarcinoma tissue. Notably, miR-30a-5p was concentrated with exosomes from the ovarian cancer cell supernatant or urine from ovarian serous adenocarcinoma patients, supporting a pathway for excretion into the urine. The results also showed that the knockdown of miR-30a-5p significantly inhibited the proliferation and migration of ovarian cancer cells. In summary, to the best of our knowledge, the present study provided the first evidence of increased miR-30a-5p in the urine of ovarian serous adeno-carcinoma patients, while the inhibition of miR-30a-5p suppressed the malignant phenotypes of ovarian cancer in vitro. Therefore, miR-30a-5p serves as a promising diagnostic and therapeutic target for ovarian serous adenocarcinoma.

Ovarian cancer is a leading cause of death from gynecologic cancers in the USA, and early diagnosis can potentially increase 5-year survival rate. Detection of biomarkers derived from hyperplasia of epithelial tissue by enzyme-linked immunosorbent assay (ELISA) proves to be a practical way of early diagnosis of ovarian cancer. However, ELISA is commonly performed in a laboratory setting, and it cannot be used in a clinical setting for on-site consultation. We have shown a microchip ELISA that detects HE4, an ovarian cancer biomarker, from urine using a cell phone integrated with a mobile application for imaging and data analysis. In microchip ELISA, HE4 from urine was first absorbed on the surface; the primary and secondary antibodies were subsequently anchored on the surface via immuno-reaction; and addition of substrate led to color development because of enzymatic labeling. The microchip after color development was imaged using a cell phone, and the color intensity was analyzed by an integrated mobile application. By comparing with an ELISA standard curve, the concentration of HE4 was reported on the cell phone screen. The presented microchip ELISA coupled with a cell phone is portable as opposed to traditional ELISA, and this method can facilitate the detection of ovarian cancer at the point-of-care (POC).

Background: Serous epithelial ovarian cancer is the most common variety of ovarian cancer and is currently diagnosed using serum CA-125 levels. HMGA1 a small 10.6-12 kDa protein, has been implicated as a potentially important tumor biomarker and may enter the urinary trace, thus potentially able to serve as a disease biomarker., Objective: To determine if urine HMGA1 can be detected and potentially serve as a clinical diagnostic biomarkers., Method: Urine was collected from 20 healthy normal control patients, 20 patients with benign gynecological disease and 55 epithelial ovarian specimens of which 20 exhibited G1/2 ovarian cancer and 35 G3 ovarian cancers. Serum was also collected from 20 healthy normal control patients and 55 serous epithelial ovarian cancers patients. HMGA1 levels were examined via enzyme-linked immunosorbent assay (ELISA) and were reported independently and normalized to urine creatinine levels. Serum CA-125 levels were examined via enzyme assay and the data was analyzed via box and ROC analysis., Results: Urine HMGA1 was significantly elevated in serous epithelial ovarian cancer specimen relative to healthy control specimens with G3 specimens exhibiting higher levels than G1-G2 specimens. ROC analysis revealed a high degree of sensitivity and specificity for urine HMGA1 detection in ovarian cancer, with a higher AUC value noted for urine HMGA1 than serum CA-125. Furthermore, urine HMGA1 and serum CA-125 combined AUC indicated that urine HMGA1 is an excellent diagnostic biomarker for serous epithelial ovarian cancer., Conclusion: Our data indicates that measuring urine HMGA1 may serve as a useful diagnostic tool.

Background: Early detection of epithelial ovarian cancer (OC) is necessary to overcome the high mortality rate of late stage diagnosis; and, examining the molecular changes that occur at early disease onset may provide new strategies for OC detection. Since the deregulation of inflammatory mediators can contribute to OC development, the purpose of this pilot study was to determine whether elevated urinary levels of Interleukin-1beta (IL-1 beta) are associated with OC and associated clinical parameters., Methods: Urinary and serum levels of IL-1 beta were analyzed by ELISA from a patient cohort consisting of healthy women (N = 10), women with ovarian benign disease (N = 23), women with OC (N = 32), women with other benign gynecological conditions (N = 22), and women with other gynecological cancers (N = 6)., Results: Average urinary IL-1 beta levels tended to be elevated in ovarian benign (1.26 pg/ml) and OC (1.57 pg/ml) patient samples compared to healthy individuals (0.36 pg/ml). Among patients with benign disease, urinary IL-1β levels were statistically higher in patients with benign inflammatory gynecologic disease compared to patients with non-inflammatory benign disease. Interestingly, urinary IL-1 beta levels tended to be 3-6x greater in patients with benign ovarian disease or OC as well as with a concomitant family history of ovarian and/or breast cancer compared to similar patients without a family history of ovarian and/or breast cancer. Lastly, there was a pattern of increased urinary IL-1 beta with increasing body mass index (BMI); patients with a normal BMI averaged urinary IL-1 beta levels of 0.92 pg/ml, overweight BMI averaged urinary IL-1 beta levels of 1.72 pg/ml, and obese BMI averaged urinary IL-1 beta levels of 5.26 pg/ml., Conclusions: This pilot study revealed that urinary levels of IL-1 beta are elevated in patients with epithelial OC supporting the thought that inflammation might be associated with cancer progression. Consequently, further studies of urinary IL-1 beta and the identification of an inflammatory profile specific to OC development may be beneficial to reduce the mortality associated with this disease.

Greater exposure to estrogens is a risk factor for ovarian cancer. To investigate the role of estrogens in ovarian cancer, a spot urine sample and a saliva sample were obtained from 33 women with ovarian cancer and 34 age-matched controls. Thirty-eight estrogen metabolites, conjugates and DNA adducts were analyzed in the urine samples using ultraperformance liquid chromatography/tandem mass spectrometry, and the ratio of adducts to metabolites and conjugates was calculated for each sample. The ratio of depurinating estrogen-DNA adducts to estrogen metabolites and conjugates was significantly higher in cases compared to controls (p < 0.0001), demonstrating high specificity and sensitivity. DNA was purified from the saliva samples and analyzed for genetic polymorphisms in the genes for two estrogen-metabolizing enzymes. Women with two low-activity alleles of catechol-O-methyltransferase plus one or two high-activity alleles of cytochrome P450 1B1 had higher levels of estrogen-DNA adducts and were more likely to have ovarian cancer. These findings indicate that estrogen metabolism is unbalanced in ovarian cancer and suggest that formation of estrogen-DNA adducts plays a critical role in the initiation of ovarian cancer., (© 2013 UICC.)

A Krukenberg tumor is a rare and potentially deadly cause of elevated serum β-hCG as part of a paraneoplastic syndrome. This study aims to describe the unusual case of a 36-year-old woman that presented to the Emergency Department (ED) with back pain and a positive urine pregnancy test. Assessment revealed no intrauterine pregnancy and a small left ovarian cyst. Further investigation showed moderately differentiated gastric adenocarcinoma with distant metastases to the spine. The patient died less than 3 months after her first presentation to the ED. Paraneoplastic syndrome, albeit rare, should be considered in the differential diagnosis of elevated β-hCG due to the high mortality associated with Krukenberg tumors.

Ovarian cancer is the type of cancer with the highest mortality rate among gynaecologic malignancies. Due to lack of screening tools, this disease is mainly diagnosed at a progressed stage, when it is too late to adequate therapy. Despite many attempts, enough sensitive and specific biomarker was not still uncovered. Fluorescence spectroscopy has proven to be a useful diagnostic tool with high efficiency. Fluorescence detection has three major advantages over other light-based investigation methods: high sensitivity, high speed, and reliability. Biological materials consist of a number of intrinsic fluorescent compounds -autofluorophores, which are associated with cardinal metabolic pathways. It is well known, that cancerous tissue metabolism is altered compared to healthy one, what influence also intrinsic fluorophores composition of bodily fluids. Urine is one of the biological fluids that could be obtained most easily and displays a blue - green fluorescence that can change in case of pathological process. Analysis of urine autofluorescence is non invasive and simple technique. Using fluorescent spectroscopy, ovarian cancer patients and healthy control group were discerned with high significance, so we predict that fluorescence analysis of urine could be a potential means of ovarian cancer screening.

Urine offers a number of attractive features as a sample type for biomarker discovery, including noninvasive sampling, quantity and availability, stability, and a narrow dynamic range. In this study we report the first application of isotope coded protein labeling (ICPL), coupled with in-solution isoelectric fractionation and LC-MALDI-TOF/TOF, to examine and prioritize urinary proteins from ovarian cancer patients. Following the definition of stringent exclusion criteria a total of 579 proteins were identified with 43% providing quantitation data. Protein abundance changes were validated for selected proteins by ESI-Qq-TOF MS, following which Western blot and immunohistochemical analysis by tissue microarray was used to explore the biological relevance of the proteins identified. Several established markers (e.g., HE4, osteopontin) were identified at increased levels in ovarian cancer patient urine, validating the approach used; we also identified a number of potential marker candidates (e.g., phosphatidylethanolamine binding protein 1, cell-adhesion molecule 1) previously unreported in the context of ovarian cancer. We conclude that the ICPL strategy for identification and relative quantitation of urine proteins is an appropriate tool for biomarker discovery studies, and can be applied for the selection of potential biomarker candidates for further characterization.

Cancer is known to induce or alter the O-glycosylation of selective proteins that may eventually be excreted in the patients' urine. The present study was performed to identify O-glycosylated proteins that are aberrantly excreted in the urine of patients with early stage ovarian cancer (OCa). These urinary glycoproteins are potential biomarkers for early detection of OCa. In this study, urinary proteins of patients with early stage OCa and age-matched OCa negative women were subjected to two-dimensional gel electrophoresis and detection using a lectin that binds to the O-glycosylated proteins. Our analysis demonstrated significant enhanced expression of clusterin and leucine-rich alpha-2-glycoprotein, but lower levels of kininogen in the urine of the OCa patients compared to the controls. The different altered levels of these urinary glycoproteins were further confirmed using competitive ELISA. Our data are suggestive of the potential use of the aberrantly excreted urinary O-glycosylated proteins as biomarkers for the early detection of OCa, although this requires further validation in a large clinically representative population.

To evaluate the application of urinary metabolomics on discovering potential biomarkers for epithelial ovarian cancer (EOC), urine samples from 40 preoperative EOC patients, 62 benign ovarian tumor (BOT) patients, and 54 healthy controls were collected and analyzed with ultraperformance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS). Good separations were obtained for EOC vs BOT, EOC vs healthy controls analyzed by partial least-squares discriminant analysis, or principal component analysis. Twenty-two ascertained metabolomic biomarkers were found to be disturbed in several metabolic pathways among EOC patients, including nucleotide metabolism (pseudouridine, N4-acetylcytidine), histidine metabolism (L-histidine, imidazol-5-yl-pyruvate), tryptophan metabolism (3-indolelactic acid), and mucin metabolism (3'-sialyllactose and 3-sialyl-N-acetyllactosamine). In addition, the concentrations of some urinary metabolites of 18 postoperative EOC patients among the 40 EOC patients changed significantly compared with those of their preoperative condition, and four of them suggested recovery tendency toward normal level after surgical operation, including N4-acetylcytidine, pseudouridine, urate-3-ribonucleoside, and succinic acid. These metabolites would be highly postulated to be associated with EOC. In conclusion, our study demonstrated that urinary metabolomics analysis by UPLC-QTOF/MS, performed in a minimally noninvasive and convenient manner, possessed great potential in biomarker discovery for EOC.

Early diagnostics of ovarian cancer is difficult, because there are no symptoms until the disease has progressed to an advanced stage. As urine contains many intrinsic fluorophores, modern fluorescence techniques are perspective candidates for new routine urine tests. The presented work deals with differences in the fluorescence of metabolites in urine of ovarian cancer patients comparing to healthy volunteers using the fluorescence excitation-emission matrices. The most serious differences were found in undiluted urine at the fluorescence emission wavelengths from 400 nm to 460 nm when excited at 310 - 390 nm. Statistical analyses of our data have shown a 5-fold reduction in the intensity of the peak at 330/420 nm (excitation/emission wavelength) for undiluted urine samples excreted by cancer patients as compared to those of normal donors. Moreover, the ratio of intensities of the peaks at 370/440 nm and at 330/420 nm is 18-times elevated in urine excreted by patients with ovarian cancer as compared to healthy urine samples. The observed changes could be interpreted as reduction of the presence of pyridoxic acid, whereas blue-fluorescing pteridines becomes dominant in excitation-emission matrices of cancer urine samples in comparison to healthy donors. We suggest pteridines, which are related to cellular metabolism, as suitable candidates for neoplasia-associated fluorescent markers in human urine. Our work showed that monitoring of human urine fluorescent metabolites offers an alternative for ovarian cancer screening.

Discovery of novel metabolite biomarker(s) for improved ovarian cancer diagnosis is of great importance. In this paper, the differences of urinary hydrophilic and hydrophobic metabolic profiling between healthy women, benign ovarian tumor, and ovarian cancer patients were studied by metabolomics strategy. Metabolites in urine were analyzed on hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) coupled to MS. Data from HILIC or RPLC, positive or negative ion detection mode were found to be complementary. Data were filtered by orthogonal signal correction (OSC) method, and the three groups were discriminated by partial least squares discrimination analysis (PLS-DA) models. By combining the four datasets, maximum information can be collected, and a PLS-DA model was built after OSC filtering. The model based on combined dataset is superior to the ones based on the separate dataset, and important metabolites were screened based on the combined dataset model. Five metabolites were found to be specific to ovarian cancer and ten metabolites were considered commonly related to ovarian cancer and benign ovarian tumor. Combination of RPLC and HILIC separation, as well as positive and negative ion detection in metabolomic studies show advantages in collecting various metabolites information that helps us better understand the metabolic event., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this study, the design, fabrication, surface functionalization and experimental characterization of an ultrasonic MEMS biosensor for urinary anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) detection with sub ng/mL sensitivity is presented. It was previously shown that urinary Bcl-2 levels are reliably elevated during early and late stages of ovarian cancer. Our biosensor uses shear horizontal (SH) surface acoustic waves (SAWs) on surface functionalized ST-cut Quartz to quantify the mass loading change by protein adhesion to the delay path. SH-SAWs were generated and received by a pair of micro-fabricated interdigital transducers (IDTs) separated by a judiciously designed delay path. The delay path was surface-functionalized with monoclonal antibodies, ODMS, Protein A/G and Pluronic F127 for optimal Bcl-2 capture with minimal non-specific adsorption. Bcl-2 concentrations were quantified by the resulting resonance frequency shift detected by a custom designed resonator circuit. The target sensitivity for diagnosis and identifying the stage of ovarian cancer was successfully achieved with demonstrated Bcl-2 detection capability of 500 pg/mL. It was also shown that resonance frequency shift increases linearly with increasing Bcl-2 concentration.

Objective: To analyze differential diagnostic accuracy of urinary human epidydimis secretory protein 4 (HE4) in patients with ovarian tumors., Materials and Methods: In the case-control study 23 patients with ovarian cancer, 37 patients with benign ovarian tumors and 18 women in the control group were included. Serum CA125 values and urinary concentrations of HE4were assessed quantitatively. Urinary creatinine concentrations and glomerular filtration rate were also determined and used to calculate ratios to HE4., Results: Higher urinary HE4 concentrations were observed in patients with late stage ovarian cancer (p=0.001) and also in patients with early stage ovarian cancer when compared to patients with benign ovarian tumors (p=0.044). On analysis where all ovarian cancer patients were included, higher diagnostic accuracy was observed with calculated ratio of HE4 to glomerular filtration rate (GFR) to unchanged urinary HE4 concentrations -AUC 0.861 vs. 0.858. When discriminatory accuracy was calculated for urinary HE4/GFR ratio and unchanged urinary HE4 concentrations, the last demonstrated a higher area under the curve - 0.701 vs. 0.602. The urinary HE4/creatinine ratio had lower discriminatory characteristics than unchanged concentrations of urinary HE4. However, HE4 serum concentration was more accurate for discrimination of patients with benign and malignant ovarian tumors when compared to urinary HE4 and CA125 in sera (AUCs were 0.868 for serum HE4 and 0.856 and 0.653 for urinary HE4 and CA125, respectively)., Conclusions: Ovarian cancer patients have higher urinary concentrations of human epidydimis secretory protein 4 than patients with benign ovarian tumors. Urinary HE4 has comparable discriminatory accuracy with serum HE4 for benign and malignant ovarian tumors and can be recommended as a non-invasive ovarian cancer risk assessment method.

Ovarian cancer is asymptomatic in the early stages and most patients present with advanced levels of disease. The lack of cost-effective methods that can achieve frequent, simple and non-invasive testing hinders early detection and causes high mortality in ovarian cancer patients. Here, we report a simple and inexpensive microchip ELISA-based detection module that employs a portable detection system, i.e., a cell phone/charge-coupled device (CCD) to quantify an ovarian cancer biomarker, HE4, in urine. Integration of a mobile application with a cell phone enabled immediate processing of microchip ELISA results, which eliminated the need for a bulky, expensive spectrophotometer. The HE4 level detected by a cell phone or a lensless CCD system was significantly elevated in urine samples from cancer patients (n = 19) than healthy controls (n = 20) (p < 0.001). Receiver operating characteristic (ROC) analyses showed that the microchip ELISA coupled with a cell phone running an automated analysis mobile application had a sensitivity of 89.5% at a specificity of 90%. Under the same specificity, the microchip ELISA coupled with a CCD had a sensitivity of 84.2%. In conclusion, integration of microchip ELISA with cell phone/CCD-based colorimetric measurement technology can be used to detect HE4 biomarker at the point-of-care (POC), paving the way to create bedside technologies for diagnostics and treatment monitoring.

Background: Small cell carcinoma (SMCC) is rarely diagnosed in urine specimens. Cytologically, this tumor is similar to pulmonary SMCC. However, clinicopathologic correlation may be required to differentiate between primary urinary bladder SMCC and metastatic SMCC from a remote primary or secondary bladder involvement by direct extension of the tumor from nearby organs (prostate, uterus, or ovary). A unique case of a rare pulmonary-type ovarian SMCC, the tumor cells of which were detected in a voided urine specimen, is described herein., Case: A 79-year-old female presented to the urologic clinic with a history of metastatic SMCC of unknown primary with hematuria. The voided urine specimen examination revealed tumor cells cytomorphologically consistent with small cell neuroendocrine carcinoma. Following cytologic diagnosis, cystoscopic examination and bladder biopsy were performed. The histopathology revealed a widely invasive tumor with a morphology typical of SMCC. The overlying urothelium was unremarkable. By immunohistochemistry, tumor cells were found positive for neuroendocrine markers, EMA and WT-1. The morphologic and immunohistochemical features of the tumor were most consistent with urinary bladder involvement by pulmonary-type primary ovarian SMCC., Conclusion: It is justified to think that SMCC cell detection in urine specimens does not necessarily imply their origin from primary bladder malignancy. Performing additional studies may be prudent in order to exclude secondary involvement of the bladder in this tumor as the correct diagnosis has significant clinical implications., (Copyright © 2011 S. Karger AG, Basel.)

Purpose: Metabolomics is a new, rapidly expanding field dedicated to the global study of metabolites in biological systems. In this article metabolomics is applied to find urinary biomarkers for breast and ovarian cancer., Experimental Design: Urine samples were collected from early- and late-stage breast and ovarian cancer patients during presurgical examinations and randomly from females with no known cancer. After quantitatively measuring a set of metabolites using nuclear magnetic resonance spectroscopy, both univariate and multivariate statistical analyses were employed to determine significant differences., Results: Metabolic phenotypes of breast and ovarian cancers in comparison with normal urine and with each other revealed significance at Bonferroni-corrected significance levels resulting in unique metabolite patterns for breast and ovarian cancer. Intermediates of the tricarboxylic acid cycle and metabolites relating to energy metabolism, amino acids, and gut microbial metabolism were perturbed., Conclusions: The results presented here illustrate that urinary metabolomics may be useful for detecting early-stage breast and ovarian cancer., (©2010 AACR.)

The main objective of the present investigation was to study the urinary neopterin excretion in the context of the activation of the adaptive cellular immune system at the tumor site. For this purpose, we compared pre-treatment urinary neopterin levels measured in 92 ovarian cancer patients, with intratumoral levels of mRNA transcripts from factors either involved in the adaptive antitumor immune defense (CD3, IFN-γ, IRF-1, IRF-2, SOCS1 and iNOS) or immune tolerance (FoxP3). This study did not reveal an association between urinary neopterin and one of these investigated "on tumor site transcripts". From all the factors reflecting the magnitude of the local adaptive antitumor response, intratumoral IRF-1 expression above the edge of the 25th percentile was found to predict most reliably favorable progression-free (median 34 months vs. 10 months; p < 0.001) and overall (median 52 months vs. 16 months; p < 0.001) survival. In contrast, pre-treatment urinary neopterin excretion above 275 μmol/mol creatinine, which indicates an unspecific activation of the innate immune system, was associated with a very poor overall survival with a median of only 11 months when compared with a median overall survival of 40 months in patients with lower urinary neopterin excretion (p = 0.021). Interestingly, the considerable survival benefit in patients with high IRF-1-expressing cancers was completely abrogated as well for progression-free as for overall survival when urinary neopterin concentrations were found to be concomitantly elevated. These findings demonstrate that in ovarian carcinomas the unspecific "cancer-related inflammation" contributes to a significant subversion of the adaptive antitumor immune defense mounted at the tumor site.

The HE4 protein is overexpressed in ovarian carcinomas and can be detected in serum by an ELISA with sensitivity similar to CA125 and higher specificity for malignant disease. We now demonstrate that HE4 can also be detected in the urine at a specificity level of 94.4%, including 13/15 (86.6%) with stage I/II and 57/64 (89.0%) with stage III/IV disease and including 90.5% of patients with serous ovarian carcinoma. Assaying serum and urine from the same patients showed similar sensitivity. Our data indicate that measuring HE4 in urine may aid diagnosis and the monitoring of response to therapy., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

Objective: The poor prognosis associated with epithelial ovarian cancer (EOC) is due to the lack of overt early symptoms and the absence of reliable diagnostic screening methods. Since many tumors over express angiogenic regulators, the purpose of this study was to determine whether elevated levels of the angiogenic or angiostatic molecules vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), endostatin (ES), and angiostatin (AS) were elevated in plasma and urine from patients with EOC., Methods: VEGF, HGF, ES and AS were assayed by ELISA in samples from pilot cohort consisting of healthy women (N=48; pre-menopausal N=23, post-menopausal N=25), women with benign gynecological disease (N=54), patients with primary peritoneal cancer (PP) (N=2) and EOC (N=35). Wherever possible, parallel serum samples were measured for CA125 levels by ELISA., Results: AS was the angioregulator that independently discriminated EOC patients from healthy individuals. Levels of urinary AS (uAS) from healthy individuals or women with benign gynecological disease averaged 21.4 ng/mL+/-3.7 and 41.5 ng/mL+/-8.8, respectively. In contrast, uAS averaged 115 ng/mL+/-39.2 and 276 ng/mL+/-45.8 from women with Stage I (N=6) and late stage (N=31) EOC, respectively. Furthermore, uAS was elevated in EOC patients regardless of tumor grade, stage, size, histological subtype, creatinine levels, menopausal status, or patient age, but appeared to complement CA125 measurements., Conclusions: Levels of AS are elevated in the urine of patients with EOC and may be of diagnostic and/or prognostic clinical importance. Further studies of uAS as a biomarker for EOC alone or in combination with other markers are warranted., (Copyright 2009 Elsevier Inc. All rights reserved.)

Purpose: The purposes of this study were to confirm previously found candidate epithelial ovarian cancer biomarkers in urine and to compare a paired serum biomarker panel and a urine biomarker panel from the same study cohort with regard to the receiver operating characteristic curve (ROC) area under the ROC curve (AUC) values., Experimental Design: Four significant urine biomarkers were confirmed among 130 pelvic mass patients in the present study. The four biomarkers form a potential urine biomarker panel. From the same study cohort, the potential urine biomarker panel was compared to a serum biomarker panel, consisting of seven proteins/peptides, OvaRI., Results: Multivariate analysis of the urine panel demonstrated a significant differentiation (p<0.0001) between epithelial ovarian cancer patients and patients with benign ovarian pelvic masses. The ROC AUC of the urine panel was 0.84 and the ROC AUC of OvaRI was 0.83. Combining the urine panel with OvaRI demonstrated a significant contribution from both, for urine peaks, OR=2.12 and for OvaRI, OR=1.39; the ROC AUC of this model was 0.88., Conclusions and Clinical Relevance: We demonstrated that both urine and serum can be used individually or in combination to potentially aid in ovarian cancer diagnostics. Urine proteomic profiling could provide biomarkers for the non-invasive test required in clinical practice., (Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose: Galectin-3 has been implicated in advanced stage of cancer disease. In the current study we examined the possibility of urinary galectin-3 levels to stage cancer disease and to follow up therapy., Experimental Design: Urine was collected from all types of cancer patients at different stages including patients undergoing radio/chemotherapy. Galectin-3 level was determined by anti-galectin-3 based ELISA and agglutination assays. Immunoblotting and purification on lactosyl affinity column further confirmed the presence of galectin-3., Results: Cancer samples exhibited stage dependent expression of galectin-3 approx. ranging from 1.0 to 3.3, 4.4 to 5.4, 5.4 to 24.7, 13.1 to 31.9, 13.9 to 32.9 ng/mg C (creatinine) for stage I-V, respectively, at P approximately <0.05 level. Galectin-3 levels were decreased by approx. threefolds after 5th day of therapy., Conclusions: Sample collection being simple and non-invasive, urinary galectin-3 may be used as a potential diagnostic tool for monitoring or follow up of the stage of cancer disease.

Background: The metabolomic approaches for mining biomarkers of women's cancers based on gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry combined with partial least squares-discriminant analysis are described., Methods: To identify urinary potential biomarkers, the qualitative and quantitative analyses were introduced with 10 breast, 9 ovarian and 12 cervical cancer patients as well as 22 normal controls, which were considered with their ages and menopausal state., Results: For comprehensive metabolomic approaches, the non-targeted qualitative profiling was first achieved to get metabolic patterns of collected samples and the targeted quantitative analysis focused on hormonal metabolism was also conducted. Two known biomarkers, i.e., 5-hydroxymethyl-2-deoxyuridine and 8-hydroxy-2-deoxyguanosine, in breast cancer were also confirmed using the present methods. In addition, 3 potential biomarkers for ovarian cancer i.e. 1-methyladenosine, 3-methyluridine, and 4-androstene-3,17-dione, which were categorized in significantly increased level using one way of variance analysis (p<0.05), were identified as quantitatively targeted metabolites with pattern analysis. The cancer markers identified in this study are highly related to metabolites which are responsible for oxidative DNA damage and DNA methylation process., Conclusion: The present metabolomic approaches are not only useful for diagnostic tools and patient stratification, but may be mapped on metabolic network to reflect disease states.

Objective(s): The poor prognosis associated with ovarian cancer is due to the lack of overt early symptoms and the absence of reliable diagnostic screening methods. Since many tumors overexpress anti-apoptotic proteins, the purpose of this study was to determine whether elevated levels of the anti-apoptotic protein Bcl-2 were present in urine from patients with ovarian cancer., Methods: Bcl-2 was assayed by ELISA in urine samples from two cohorts consisting of a total of 77 healthy women, 161 women with benign gynecologic disease and 150 women with ovarian cancer, 13 with early and 137 with late stage disease, respectively. Wherever possible, parallel serum samples were measured for CA125 levels by ELISA., Results: Urinary levels of Bcl-2 from healthy individuals or women with benign disease averaged 0.59 ng/ml+/-0.61 and 1.12 ng/ml+/-0.79, respectively. In contrast, urinary levels of Bcl-2 averaged 2.60 ng/ml+/-2.23 and 3.58 ng/ml+/-1.55 from women with early (N=13) and late (N=137) stage ovarian cancer. Further, urinary levels of Bcl-2 were elevated in ovarian cancer patients regardless of tumor grade, stage, size, histologic subtype, creatinine levels or patient age, but appeared to complement CA125 measurements., Conclusion(s): Levels of Bcl-2 are elevated in the urine of patients with ovarian cancer and may be of diagnostic and/or prognostic clinical importance. Further studies of urinary Bcl-2 as a biomarker for ovarian cancer alone or in combination with other markers are warranted.

Objective: To examine whether urine can be used to measure specific ovarian cancer proteomic profiles and whether one peak alone or in combination with other peaks or CA125 has the sensitivity and specificity to discriminate between ovarian cancer pelvic mass and benign pelvic mass., Methods: A total of 209 women were admitted for surgery for pelvic mass at the Gynaecological Department at Rigshospitalet, Copenhagen. Of the women, 156 had benign gynaecological tumors, 13 had borderline tumors and 40 had malignant epithelial ovarian cancer. The prospectively and preoperatively collected urine samples were aliquotted and frozen at -80 degrees until the time of analysis. The urine was fractionated using equalizer bead technology and then analyzed with surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. Biomarkers were purified and identified using combinations of chromatographic techniques and tandem mass spectrometry., Results: Benign and malignant ovarian cancer cases were compared; 21 significantly different peaks (p<0.001) were visualized using Mann-Whitney analysis, ranging in m/z values from 1,500 to 185,000. The three most significant peaks were purified and identified as fibrinogen alpha fragment (m/z=2570.21), collagen alpha 1 (III) fragment (m/z=2707.32) and fibrinogen beta NT fragment (m/z=4425.09). The area under the receiver operator characteristic curve (ROC AUC) value for these three peaks in combination was 0.88, and their ROC AUC value in combination with CA125 was 0.96., Conclusion: This result supports the feasibility of using urine as a clinical diagnostic medium, and the ROC AUC value for the three most significant peaks in combination with or without CA125 demonstrates the enhanced prediction performance of combined marker analysis.