Eva Jandáková

Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer

Flow cytometry is a powerful method, which is widely used for high-throughput quantitative and qualitative analysis of cells. However, its straightforward applicability for extracellular vesicles (EVs) and mainly exosomes is hampered by several challenges, reflecting mostly the small size of these vesicles (exosomes: ~80–200 nm, microvesicles: ~200–1,000 nm), their polydispersity, and low refractive index. The current best and most widely used protocol for beads-free flow cytometry of exosomes uses ultracentrifugation (UC) coupled with floatation in sucrose gradient for their isolation, labeling with lipophilic dye PKH67 and antibodies, and an optimized version of commercial high-end cytometer for analysis. However, this approach requires an experienced flow cytometer operator capable of manual hardware adjustments and calibration of the cytometer. Here, we provide a novel and fast approach for quantification and characterization of both exosomes and microvesicles isolated from cell culture media as well as from more complex human samples (ascites of ovarian cancer patients) suitable for multiuser labs by using a flow cytometer especially designed for small particles, which can be used without adjustments prior to data acquisition. EVs can be fluorescently labeled with protein-(Carboxyfluoresceinsuccinimidyl ester, CFSE) and/or lipid- (FM) specific dyes, without the necessity of removing the unbound fluorescent dye by UC, which further facilitates and speeds up the characterization of microvesicles and exosomes using flow cytometry. In addition, double labeling with protein- and lipid-specific dyes enables separation of EVs from common contaminants of EV preparations, such as protein aggregates or micelles formed by unbound lipophilic styryl dyes, thus not leading to overestimation of EV numbers. Moreover, our protocol is compatible with antibody labeling using fluorescently conjugated primary antibodies. The presented methodology opens the possibility for routine quantification and characterization of EVs from various sources. Finally, it has the potential to bring a desired level of control into routine experiments and non-specialized labs, thanks to its simple bead-based standardization.

L1CAM expression in endometrial carcinomas: an ENITEC collaboration study

Background:Identification of aggressive endometrioid endometrial carcinomas (EECs) and non-endometrioid carcinomas (NEECs) is essential to improve outcome. L1 cell adhesion molecule (L1CAM) expression is a strong prognostic marker in stage I EECs, but less is known about L1CAM expression in advanced-stage EECs and NEECs. This study analyses L1CAM expression in a clinically representative cohort of endometrial carcinomas.Methods:The expression of L1CAM was immunohistochemically determined in 1199 endometrial carcinomas, treated at one of the European Network for Individualized Treatment of Endometrial Cancer (ENITEC) centres. Staining was considered positive when >10% of the tumour cells expressed L1CAM. The association between L1CAM expression and several clincopathological characteristics and disease outcome was calculated.Results:In all, L1CAM was expressed in 10% of the 935 stage I EECs, 18% of the 160 advanced stage EECs, and 75% of the 104 NEECs. The expression of L1CAM was associated with advanced stage, nodal involvement, high tumour grade, non-endometrioid histology, lymphovascular space invasion, and distant recurrences in all cases, and with reduced survival in the EECs, but not in the NEECs.Conclusions:The expression of L1CAM is a strong predictor of poor outcome in EECs, but not NEECs. It is strongly associated with non-endometrioid histology and distant spread, and could improve the postoperative selection of high-risk endometrial carcinomas. The value of L1CAM expression in the preoperative selection of high-risk endometrial carcinomas should be studied.

Evaluation of Cell-Free Urine microRNAs Expression for the Use in Diagnosis of Ovarian and Endometrial Cancers. A Pilot Study

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.

Electrochemical chip-based genomagnetic assay for detection of high-risk human papillomavirus DNA.

Cervical cancer, being the fourth leading cause of cancer death in women worldwide, predominantly originates from a persistent infection with a high-risk human papillomavirus (HPV). Detection of DNA sequences from these high-risk strains, mostly HPV-16 and HPV-18, represents promising strategy for early screening, which would help to identify women with higher risk of cervical cancer. In developing countries, inadequate screening options lead to disproportionately high mortality rates, making a fast and inexpensive detection schemes highly important. Electrochemical sensors and assays offer an alternative to current methods of detection. We developed an electrochemical-chip based assay, in which target HPV DNA is captured via magnetic bead-modified DNA probes, followed by an antidigoxigenin-peroxidase detection system at screen-printed carbon electrode chips, enabling parallel measurements of eight samples simultaneously. We show sensitive detection in attomoles of HPV DNA, selective discrimination between HPV-16 and HPV-18 and good reproducibility. Most importantly, we show application of the assay into both cancer cell lines and cervical smears from patients. The electrochemical results correlated well with standard methods, making this assay potentially applicable in clinical practice.

Prognostic value of human epididymis protein 4 in endometrial cancer and its utility for surgical staging

An optimal surgical staging in the group of patients with the high‐risk type of endometrial cancer is often limited by age and serious internal comorbidities. Therefore, in this study we focused on human epididymis protein 4 and its contribution to the preoperative differentiation of prognostically distinct groups of patients and to individualized surgical treatment as compared with cancer antigen (CA) 125 and imaging methods.

Added Value of Estrogen Receptor, Progesterone Receptor, and L1 Cell Adhesion Molecule Expression to Histology-Based Endometrial Carcinoma Recurrence Prediction Models: An ENITEC Collaboration Study

Objectives Endometrial carcinoma mortality is mainly caused by recurrent disease, and various immunohistochemical markers to predict recurrences have been studied. Loss of the estrogen receptor (ER) and progesterone receptor (PR) and the presence of the L1 cell adhesion molecule (L1CAM) are promising markers, but their combined value has not been studied. Materials and Methods Expression of ER, PR, and L1CAM was immunohistochemically determined in 293 endometrial carcinomas from 11 collaborating European Network for Individualized Treatment of Endometrial Cancer centers. Estrogen receptor, PR, or L1CAM staining was considered positive or negative when expressed by greater than or equal to 10% or less than 10% of the tumor cells, respectively. The association between these markers and clinicopathological markers, and their combined value in predicting survival were calculated, both in the entire cohort and in a selected groups of stage I endometrioid and low-risk stage I endometrioid carcinomas. Results Estrogen receptor and PR were negative in 19% and 28% of the cases, respectively, and L1CAM was positive in 18%. All 3 were associated with advanced stage, high-grade, nonendometrioid histology, lymphovascular space invasion (LVSI), and reduced disease-free survival. Only advanced stage, loss of PR, and LVSI were associated with reduced disease-free survival in multivariate analysis. A prognostic model including these 3 markers was superior to 1 including only the 3 immunohistochemical markers, which was superior to the traditional model. In both the stage I endometrioid and the low-risk stage I endometrioid groups, only loss of PR was associated with reduced disease-free survival. Conclusions Loss of ER and PR, and the presence of L1CAM are associated with high risk characteristics, and loss of PR is the strongest predictor of recurrent disease. Although a combination of these 3 markers is slightly superior to the traditional histological markers, a prognostic model including stage, PR expression, and LVSI is the most promising model in the identification of high risk carcinomas. In the stage I endometrioid carcinomas, PR immunohistochemistry appears to be of additional value in predicting recurrences.

Ascites-Derived Extracellular microRNAs as Potential Biomarkers for Ovarian Cancer

Ovarian cancer as the most fatal gynecological malignancy is often manifested by excessive fluid accumulation known as ascites or effusion. Ascites-derived microRNAs (miRNAs) may be closely associated with ovarian cancer progression. However, our knowledge of their roles, altered expression, and clinical outcomes remained limited. In this study, large-scale expression profiling of 754 human miRNAs was performed using real-time quantitative polymerase chain reaction and 384-well TaqMan array human miRNA A and B cards to identify differentially expressed miRNAs between extracellular fraction of the ascitic fluid associated with high-grade serous ovarian carcinomas and control plasma. Of the 754 miRNAs, 153 were significantly differentially expressed relative to the controls. Expression of 7 individual miRNAs (miR-200a, miR-200b, miR-200c, miR-141, miR-429, miR-1290, and miR-30a-5p) was further validated in extended sample sets, including serous, endometrioid, and mucinous subtypes. All miR-200 family members and miR-1290 were conspicuously overexpressed, while miR-30a-5p was only weakly overexpressed. The ability of miRNAs expression to discriminate the pathological samples from the controls was strong. Receiver operating characteristic curve analyses found area under the curve (AUC) values of 1.000 for miR-200a, miR-200c, miR-141, miR-429, and miR-1290 and of AUC 0.996 and 0.885 for miR-200b and miR-30a-5p, respectively. Preliminary survival analyses indicated low expression level of miR-200b as significantly related to longer overall survival (hazard ratio [HR]: 0.25, mean survival 44 months), while high expression level was related to poor overall survival (HR: 4.04, mean survival 24 months). Our findings suggested that ascites-derived miRNAs should be further explored and evaluated as potential diagnostic and prognostic biomarkers for ovarian cancer.

Cell-Free Urinary MicroRNAs Expression in Small-Scale Experiments

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.

Modified posterior pelvic exenteration for advanced ovarian malignancies: a single-institution study of 35 cases

This study aimed to investigate the possible benefits of a complete cytoreduction in patients with advanced ovarian cancer and concomitant rectal invasion. Furthermore, we evaluated the morbidity associated with radical surgery.

Patient with inoperable pheochromocytoma

Malignant pheochromocytoma is a tumour with a very low incidence that occurs sporadically or in the presence of multiple endocrine neoplasia. We present the case of a woman with a sporadic occurrence of pheochromocytoma diagnosed in the phase of multiple dissemination in the abdominal cavity and overexpressing adrenaline, noradrenaline, and dopamine. Local transarterial chemoembolization and systemic treatment with lanreotide resulted in a very good response, a decrease in the production of catecholamines for 12 months and a partial decrease for another 8 months, with stabilization of disease determined by imaging. Systemic treatment with tegafur resulted in disease stabilization lasting 50 months, after which the drug was discontinued because of adverse effects. Maintenance therapy with lanreotide continues, and no disease progression has been observed for 4 months. The treatment algorithm for such patients is multidisciplinary and must always take into account the current scope of the disease, intercurrence, and the general condition of the patient.