Vít Weinberger

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.

A prospective study examining the incidence of asymptomatic and symptomatic lymphoceles following lymphadenectomy in patients with gynecological cancer

OBJECTIVE To identify the incidence of asymptomatic and symptomatic (i.e., causing pain, hydronephrosis, venous thrombosis, acute lymphedema of the lower or urinary urgency) lymphoceles, as well as risk factors for their development, through a prospective study of patients undergoing sole pelvic or combined pelvic and paraaortic lymphadenectomy for gynecological cancer. METHODS Patients with endometrial, ovarian or cervical cancer scheduled for sole pelvic or combined pelvic and paraaortic lymphadenectomy as a primary surgical treatment or salvage surgery for recurrence were enrolled at single institution from February 2006 to November 2010 and prospectively followed up with ultrasound. RESULTS Of 800 patients who underwent sole pelvic or combined pelvic and paraaortic lymphadenectomy for gynecological cancer, the overall incidence of lymphoceles was 20.2%, with symptomatic lymphoceles occurring in 5.8% of all patients. Lymphoceles are predominantly located on the left pelvic side wall. Lymphadenectomy in ovarian cancer, a higher number of lymph nodes obtained (>27), and radical hysterectomy in cervical cancer were found to be independent risk factors for the development of symptomatic lymphoceles. CONCLUSIONS The overall incidence of lymphocele development after lymphadenectomy for gynecological cancer remains high. However, the majority of lymphoceles are only incidental finding without clinical impact. A symptomatic lymphocele is an uncommon event, occurring in only 5.8% of patients. Symptomatic lymphoceles tend to develop earlier than asymptomatic. Although such risk factors are hard to avoid, patients known to be at an increased risk of developing symptomatic lymphoceles can be counseled appropriately and followed up for specific symptoms relating to lymphocele development.

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.

Lymphocele: prevalence and management in gynecological malignancies

A lymphocele is a cystic mass that may occur in the retroperitoneum following a systematic pelvic and/or para-aortic lymphadenectomy. Lymphoceles may be the cause of severe morbidity, or rarely mortality. Symptomatic lymphoceles manifest with pain, compression of adjacent structures, lymphoedema, deep vein thrombosis or inflammation. The morbidity associated with a symptomatic lymphocele may reduce the quality of life of a patient, as well as delay subsequent cancer treatment. The number and positivity of removed lymph nodes, surgical approach, type of tumor, radiotherapy and BMI rate are among the most discussed risk factors of lymphocele formation. The incidence of postoperative lymphocele is reported in the broad range of 1–58%; 5–18% of those who are symptomatic. Only symptomatic lymphoceles should be treated. Mini-invasive methods involving catheter drainage and sclerotization tend to prevail. Surgery either via laparoscopy or laparotomy remains an option in recurring, poorly accessible or inflammatory lymphoceles.

Serous tubal intraepithelial carcinoma (STIC) – clinical impact and management

ABSTRACT Introduction: Serous tubal intraepithelial carcinoma (STIC) is most likely precursor lesion of the most part of high-grade serous pelvis carcinomas, carcinosarcoma and undifferentiated carcinoma with incidence of 0.6% to 7% in BRCA carriers or women with strong family history of breast or ovarian carcinoma. STIC is a pathomorphologically and immunohistochemically detectable lesion which biological significance and clinical relevance is unknown. Areas covered: We investigate methods of STIC diagnostics and we present an overview of recent studies and available knowledge on surgical management, adjuvant chemotherapy and subsequent follow-up procedure in women with an isolated STIC. Expert commentary: Patients found to have an incidental STIC lesion should be referred for screening of BRCA1/2 mutation. In absence of an invasive disease, follow-up of patient remains a reasonable choice. A rational scheme should include check-ups every 6 months consisting of gynecological examinations, CA 125 and/or HE4 and pelvic ultrasound examination by an expert sonographer.

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.

Secondary cytoreductive surgery - viable treatment option in the management of platinum-sensitive recurrent ovarian cancer

OBJECTIVE This study aimed to evaluate the impact of the secondary cytoreductive surgery on survival parameters in women with platinum-sensitive recurrent ovarian cancer who undergone secondary cytoreduction following chemotherapy compared to women who recieved chemotherapy alone. STUDY DESIGN In a retrospective study, data were rewieved from women who were diagnosed and treated with ovarian cancer and its primary platinum-sensitive recurrence at the University Hospital Brno in the Czech Republic between November 2009 and March 2016. Out of the total number of 62 patients with recurrence, 30 women underwent cytoreductive surgery plus chemotherapy and 32 were treated with chemotherapy alone. The good performance status expressed by ECOG score 0-1, the single site of recurrence regardless of platinum-free interval or multiple sites of recurrence but no carcinomatosis and platinum-free interval >12 months, and no or small-volume ascites (<500 ml) were considered inclusion criteria for cytoreductive surgery. Women not meeting these criteria were treated by chemotherapy alone. Descriptive statistics, Kaplan-Meier survival curves and Log-Rank test were used for statistical estimations. RESULTS The analysis confirmed more favorable prognosis in patient group treated with a combination secondary cytoreduction and chemotherapy. Mean disease-free survival (DFS) was 49.8 months (95% CI; 33.2-66.3) and mean overall survival (OS) stood at 54.0 months (95% CI; 39.4-68.6) in this patient cohort, while in patient group treated with chemotherapy alone it was found that mean DFS was 16.6 months (95% CI; 7.4-25.8) and mean OS stood at 26.2 months (95% CI; 16.6-35.8). When testing the difference between survival curves, statistically significant differences were observed in both DFS (p = 0.010) and OS (p = 0.007) rates between two treatment groups. Age < 60 years at the time of recurrence and zero macroscopic residual disease after secondary cytoreduction were identified as favorable prognostic factors for both DFS and OS in a multivariate analysis. CONCLUSION Secondary cytoreductive surgery is acceptable as a viable treatment option for highly selected women with ovarian cancer recurrence. Complete resection is considered ultimate goal of secondary cytoreduction on condition that the balance between maximal survival gain and minimal operative morbidity will be kept.

Accuracy of ultrasound in prediction of rectosigmoid infiltration in epithelial ovarian cancer

To examine prospectively the accuracy of ultrasound in predicting rectosigmoid tumor infiltration in patients with epithelial ovarian cancer.