Serum Proteomic Profiles of Patients with High and Low Risk of Endometrial Cancer Recurrence.

Endometrial cancer is the most common gynecological cancer worldwide. Classifying endometrial cancer into low- or high-risk groups based on the following features is recommended: tumor grade, lymphovascular space invasion, myometrial involvement, and non-endometrioid histology. Despite the recent progress in molecular profiling of endometrial cancer, a substantial group of patients are misclassified based on the current criteria. This study aimed to identify proteins that could be used as biomarkers for the stratification of endometrial cancer patients into low- or high-risk groups. The proteomic analysis of serum samples from endometrial cancer patients was performed using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). The data were then analyzed using chemometric algorithms to identify potential biomarkers. Nineteen precursor ions were identified as fragments of eighteen proteins which included (1) connective tissue matrix proteins, (2) cytoskeletal proteins, and (3) innate immune system molecules and stress proteins. These biomarkers could be used to stratify the high- and low-risk patients, thus enabling more precise treatment decisions.

Cancer Stem Cell Markers-Clinical Relevance and Prognostic Value in High-Grade Serous Ovarian Cancer (HGSOC) Based on The Cancer Genome Atlas Analysis.

Cancer stem cells (CSCs) may contribute to an increased risk of recurrence in ovarian cancer (OC). Further research is needed to identify associations between CSC markers and OC patients' clinical outcomes with greater certainty. If they prove to be correct, in the future, the CSC markers can be used to help predict survival and indicate new therapeutic targets. This study aimed to determine the CSC markers at mRNA and protein levels and their association with clinical presentation, outcome, and risk of recurrence in HGSOC (High-Grade Serous Ovarian Cancer). TCGA (The Cancer Genome Atlas) database with 558 ovarian cancer tumor samples was used for the evaluation of 13 CSC markers (ALDH1A1, CD44, EPCAM, KIT, LGR5, NES, NOTCH3, POU5F1, PROM1, PTTG1, ROR1, SOX9, and THY1). Data on mRNA and protein levels assessed by microarray and mass spectrometry were retrieved from TCGA. Models to predict chemotherapy response and survival were built using multiple variables, including epidemiological data, expression levels, and machine learning methodology. ALDH1A1 and LGR5 mRNA expressions indicated a higher platinum sensitivity (p = 3.50 × 10-3; p = 0.01, respectively). POU5F1 mRNA expression marked platinum-resistant tumors (p = 9.43 × 10-3). CD44 and EPCAM mRNA expression correlated with longer overall survival (OS) (p = 0.043; p = 0.039, respectively). THY1 mRNA and protein levels were associated with worse OS (p = 0.019; p = 0.015, respectively). Disease-free survival (DFS) was positively affected by EPCAM (p = 0.004), LGR5 (p = 0.018), and CD44 (p = 0.012). In the multivariate model based on CSC marker expression, the high-risk group had 9.1 months longer median overall survival than the low-risk group (p < 0.001). ALDH1A1, CD44, EPCAM, LGR5, POU5F1, and THY1 levels in OC may be used as prognostic factors for the primary outcome and help predict the treatment response.

Granzyme B in peripheral blood mononuclear cells as a measure of cell-mediated immune response in paraneoplastic neurological syndromes and malignancy.

BACKGROUND: Paraneoplastic neurological syndromes (PNS) may coexist with ovarian or lung cancers. Some tumors coexisting with PNS are smaller and have a better prognosis than tumors without PNS. PNS may constitute an opportunity to observe a natural immune antitumor response. We aimed to investigate a cytotoxic immune response by measuring granzyme B (GrB) in peripheral blood mononuclear cells (PBMC) in patients affected with ovarian or lung malignancy, with and without accompanying PNS. METHODS: We enrolled patients with: nonmalignant lesions (n = 21), ovarian cancer (n = 19), lung cancer (n = 57), and PNS (n = 30). PBMC were isolated by density gradient centrifugation with Ficoll-Paque. We evaluated the expression of GrB in PBMC lysates by ELISA and normalized to protein content as measured by the Lowry method. RESULTS: GrB levels in PBMC in the group with malignant tumors-median 1650 pg/mg protein (interquartile range 663-3260 pg/mg) and in patients with PNS-median 1890 pg/mg protein (range 1290-2640 pg/mg) was lower than in control group with nonmalignant lesions-median 5240 pg/mg protein (range 2160-7440 pg/mg), p = 0.0003 and p = 0.0038, respectively. The differences in GrB levels in PBMC between these groups were independent of epidemiological factors-age, sex, body mass index (BMI), and the number of immune cells, as confirmed by multiple regression analysis. Within the group of patients with malignancy and PNS, GrB levels in PBMC were elevated if onconeural antibodies were detected (2610; 2390-3700 pg/mg protein) as compared to patients without antibodies (1680; 970-1880 pg/mg protein, p = 0.035). GrB in PBMC was higher if the malignancy was diagnosed at the low (3060; 2120-5220 pg/mg protein) as compared to the high stage (1330; 348-2140, p = 0.00048). In patients with lung cancer, the expression of GrB in PBMC was lower (1430; 635-2660 pg/mg protein) than in the group with ovarian cancer (2580; 1730-3730, p = 0.02). CONCLUSION: The cytotoxic response measured in peripheral blood by GrB in PBMC is impaired both in the course of malignancy and PNS. Levels of GrB in PBMC were higher if onconeural antibodies were detected. Tracking reactive immune responses, such as GrB in PBMC may have diagnostic and monitoring value in malignancy and PNS.

Modeling of Intracellular Taurine Levels Associated with Ovarian Cancer Reveals Activation of p53, ERK, mTOR and DNA-Damage-Sensing-Dependent Cell Protection.

Taurine, a non-proteogenic amino acid and commonly used nutritional supplement, can protect various tissues from degeneration associated with the action of the DNA-damaging chemotherapeutic agent cisplatin. Whether and how taurine protects human ovarian cancer (OC) cells from DNA damage caused by cisplatin is not well understood. We found that OC ascites-derived cells contained significantly more intracellular taurine than cell culture-modeled OC. In culture, elevation of intracellular taurine concentration to OC ascites-cell-associated levels suppressed proliferation of various OC cell lines and patient-derived organoids, reduced glycolysis, and induced cell protection from cisplatin. Taurine cell protection was associated with decreased DNA damage in response to cisplatin. A combination of RNA sequencing, reverse-phase protein arrays, live-cell microscopy, flow cytometry, and biochemical validation experiments provided evidence for taurine-mediated induction of mutant or wild-type p53 binding to DNA, activation of p53 effectors involved in negative regulation of the cell cycle (p21), and glycolysis (TIGAR). Paradoxically, taurine's suppression of cell proliferation was associated with activation of pro-mitogenic signal transduction including ERK, mTOR, and increased mRNA expression of major DNA damage-sensing molecules such as DNAPK, ATM and ATR. While inhibition of ERK or p53 did not interfere with taurine's ability to protect cells from cisplatin, suppression of mTOR with Torin2, a clinically relevant inhibitor that also targets DNAPK and ATM/ATR, broke taurine's cell protection. Our studies implicate that elevation of intracellular taurine could suppress cell growth and metabolism, and activate cell protective mechanisms involving mTOR and DNA damage-sensing signal transducti.

Cell-mediated immune responses in paraneoplastic neurological syndromes.

Paraneoplastic neurological syndromes (PNS) are disorders of the nervous system that are associated with remote effects of malignancy. PNS are considered to have an autoimmune pathology. It has been suggested that immune antitumor responses are the origin of improved outcome in PNS. We describe cell-mediated immune responses in PNS and their potential contributions to antitumor reactions. Experimental and neuropathological studies have revealed infiltrates in nervous tissue and disturbances in lymphocyte populations in both cerebrospinal fluid and peripheral blood. A predominance of cytotoxic T lymphocytes (CTLs) over T helper cells has been observed. CTLs can be specifically aggressive against antigens shared by tumors and nervous tissue. Based on genetic studies, a common clonal origin of lymphocytes from blood, tumor, and nervous tissue is suggested. Suppressive regulatory T (Treg) lymphocytes are dysfunctional. Simultaneously, in tumor tissue, more intense cell-mediated immune responses are observed, which often coincide with a less aggressive course of neoplastic disease. An increased titer of onconeural antibodies is also related to better prognoses in patients without PNS. The evaluation of onconeural and neuronal surface antibodies was recommended in current guidelines. The link between PNS emergence and antitumor responses may result from more active CTLs and less functional Treg lymphocytes.

Choriocarcinoma complicated with intra-abdominal and intrapleural hemorrhage in pregnancy - case report.

Background: Choriocarcinoma is a rare neoplasm, exceptionally uncommon during an ongoing pregnancy. The disease often has a metastatic character, causing severe symptoms from various anatomic sites like the lungs, central nervous system, vagina, pelvis, or liver. Due to the condition's rarity, evidence on how to treat the choriocarcinoma originating during pregnancy remains scarce. Case presentation: Here, we present a case of a patient who developed choriocarcinoma before the 29th week of gestation. The neoplasm had a metastatic character, resulting in hemorrhage complicated by a hypovolemic shock. The patient underwent an emergency cesarean section and several surgeries to stop the massive hemorrhage. The treatment of the choriocarcinoma included chemotherapy with methotrexate followed by an EMA-CO regimen. The patient had a complete response to the therapy. The neonate suffered from complications related to prematurity. Conclusion: Metastatic choriocarcinoma can be a diagnostic and therapeutic challenge during ongoing pregnancy. Treatment of the disease can be associated with severe complications, but a complete response to chemotherapy is possible with a favorable long-term prognosis.

Immunotherapy in Ovarian Cancer.

Despite advances in surgery and chemotherapy, ovarian cancer remains one of the most lethal malignancies. Hence, the implementation of novel treatment approaches is required to improve the outcomes of the disease. Immunotherapy has been proven to be effective in many tumors and has already been incorporated into clinical practice. In this review, we describe key strategies in immunotherapy of ovarian cancer and summarize data from clinical studies assessing immunological prospects which could improve ovarian cancer treatment approaches in the future. The most notable current strategies include checkpoint blockade agents, the use of vaccines, adoptive cell transfer, as well as various combinations of these methods. While several of these options are promising, large controlled randomized studies are still needed to implement new immunotherapeutic options into clinical practice.

New Kid on the Block: The Efficacy of Phytomedicine Extracts Urox® in Reducing Overactive Bladder Symptoms in Rats.

The aim of the current study was to determine if phytomedicine (Urox®) would reverse retinyl acetate (RA)-induced changes characteristic of bladder overactivity. There were 60 rats divided into the following 4 groups: I-control, II-received RA to induce detrusor overactivity (DO), III-received Urox (840 mg daily for 14 days), and IV-received combination of RA and Urox®. The cystometry was performed 2 days after the last dose of Urox®. Next, urothelium thickness and biochemical parameter measurements were performed. In group IV, a decrease in basal pressure and detrusor overactivity index was noted when compared to group II. Furthermore, in group IV the following parameters were increased: threshold pressure, voided volume, intercontraction interval, and bladder compliance in comparison with group II. There were significant elevations in c-Fos expression in the neuronal voiding centers in group II, while the expression of c-Fos in group IV was normalized. No significant changes in the values of the analyzed biomarkers in group III were found, while in group II, an elevation in BDNF, NGF, CGRP, ATP, Rho kinase, malondialdehyde, 3-nitrotyrosine, TRPV1, OCT-3, and VAChT and then a decrease in E-cadherin and Z01 were found. A successful restoration of all the abovementioned biomarkers' levels was observed in group IV. Phytomedicine extracts (Urox®) were found to be potent in reversing RA-induced changes in several cystometric and biochemical parameters that are determinants of overactive bladder (OAB). The actions of Urox® were proved to be dependent on several factors, such as growth factors and several OAB biomarkers but not pro-inflammatory cytokines.

Serum homocysteine and vitamin B12 levels in women with gestational diabetes mellitus.

OBJECTIVES: Gestational diabetes mellitus (GDM) is described as a glucose intolerance of variable severity which begun or was firstly recognized during gravidity. Two major metabolic disorders, insulin resistance and ß-cell dysfunction, currently play major role in pathogenesis of GDM. Our intention was to investigate total serum homocysteine and vitamin B12 levels in pregnant women with GDM and non-diabetic gravid women. MATERIAL AND METHODS: Serum homocysteine and vitamin B12 levels were prospectively measured in a total of 79 pregnant women, 60 of whom were diagnosed with GDM, and 19 of whom were healthy controls. Serum homocysteine levels were analyzed by ELISA. Vitamin B12 concentrations were determined by chemiluminescent immunoassay, and lipids were determined enzymatically. RESULTS: GDM and control groups did not differ in terms of the serum homocysteine levels (median 7.24 vs 7.97 umol/L, respectively, p = 0.15). Nor did we find any association between serum homocysteine levels and BMI (r = 0.06, p = 0.55, respectively). There was no correlation between serum homocysteine and fasting serum glucose (r = 0.3, p = 0.8, respectively). There was no relationship between serum homocysteine concentrations and glycosylated hemoglobin (HgbA1c) levels (r = 0.06, p = 0.67, respectively). Serum vitamin B12 concentrations did not differ between the GDM and control groups (median 286 vs 262 pg/mL, respectively, p = 0.17). We found that levels of Vitamin B12 correlated inversely with fasting serum glucose concentrations (r = -0.44, p = 0.0009). Vitamin B12 concentrations increased along with LDL (r = 0.27, p = 0.043) and HDL (r = 0.38, p = 0.004) levels, however were inversely correlated with serum triglycerides (r = -0.34, p = 0.009). CONCLUSIONS: GDM patients with low Vitamin B12 values tend to have higher fasting serum glucose and altered lipid profiles (high triglycerides, low HDL and LDL). In women with GDM, serum homocysteine levels are not associated with HbA1c level, fasting glycemia, or BMI.

The value of an initial drop in human Chorionic gonadotropin levels in predicting a response to methotrexate in women with low-risk gestational trophoblastic neoplasia.

OBJECTIVES: The early identification of patients who are being treated for low-risk gestational trophoblastic neoplasia (LRGTN) with single-agent chemotherapy, who are at high risk of developing chemoresistance, is of crucial importance. The aim of our research was to evaluate the pretreatment beta subunit of human chorionic gonadotropin (ßhCG) concentration and its decrease after the administration of the first course of methotrexate (MTX) in predicting later chemo-resistance to single-agent chemotherapy. MATERIAL AND METHODS: A total of 46 patients diagnosed with LRGTN treated with a 5-day methotrexate (MTX) regimen were retrospectively studied. 24 of the patients were successfully cured with only MTX therapy (MTX group). The disease was considered resistant in the remaining 22 patients who, after MTX therapy, required further chemotherapy with an EMA/CO regimen (EMA/CO group). To compare changes in the ßhCG concentrations between the two courses of treatment (and the two groups), we calculated the percentage of decline. We determined the specificity and sensitivity of the initial ßhCG level and its percentage decline, as a potential predictor of the need for a future EMA/CO regimen. For diagnostic purposes, ßhCG levels were measured before the first and second administrations of MTX with a commercial ELISA kit. RESULTS: In the EMA/CO group, we found the initial ßhCG level before the first MTX dose was higher (median = 6275 mIU/mL, range: 21.53-192.610.0 mIU/mL) than in the MTX group (median = 532 mIU/mL, range: 56.5 mIU/mL-360.397.0 mIU/mL) (p = 0.034, Mann-Whitney test). The percentage decreases in the ßhCG values relative to the initial concentrations were higher in the MTX group (median decrease = 82.7%, range: from 13.3% to 99.9%) than in the EMA/CO group (median de- crease = 71.1%, range: from an increase of 56.1% to a decrease of 97.1%) (p = 0.0079, Mann-Whitney test). An analysis of the ROC curves implied optimal cutoff values for the initial ßHCG (6054 IU, sensitivity = 55%, specificity = 86%) and the percentage change in ßhCG levels (decrease by 76.5%, sensitivity = 72%, specificity = 71%). CONCLUSIONS: Women with initially higher ßhCG levels have a greater risk of developing MTX chemo resistance. It would be advantageous to consider administering an EMA/CO regimen in women with LRGTN when their initial ßhCG levels are greater than 6000.

Membrane-bound Gaussia luciferase as a tool to track shedding of membrane proteins from the surface of extracellular vesicles.

Extracellular vesicles (EVs) released by cells play a role in intercellular communication. Reporter and targeting proteins can be modified and exposed on the surface of EVs to investigate their half-life and biodistribution. A characterization of membrane-bound Gaussia luciferase (mbGluc) revealed that its signal was detected also in a form smaller than common EVs (<70 nm). We demonstrated that mbGluc initially exposed on the surface of EVs, likely undergoes proteolytic cleavage and processed fragments of the protein are released into the extracellular space in active form. Based on this observation, we developed a new assay to quantitatively track shedding of membrane proteins from the surface of EVs. We used this assay to show that ectodomain shedding in EVs is continuous and is mediated by specific proteases, e.g. metalloproteinases. Here, we present a novel tool to study membrane protein cleavage and release using both in vitro and in vivo models.

Methods for Systematic Identification of Membrane Proteins for Specific Capture of Cancer-Derived Extracellular Vesicles.

Analysis of cancer-derived extracellular vesicles (EVs) in biofluids potentially provides a source of disease biomarkers. At present there is no procedure to systematically identify which antigens should be targeted to differentiate cancer-derived from normal host cell-derived EVs. Here, we propose a computational framework that integrates information about membrane proteins in tumors and normal tissues from databases: UniProt, The Cancer Genome Atlas, the Genotype-Tissue Expression Project, and the Human Protein Atlas. We developed two methods to assess capture of EVs from specific cell types. (1) We used palmitoylated fluorescent protein (palmtdTomato) to label tumor-derived EVs. Beads displaying antibodies of interest were incubated with conditioned medium from palmtdTomato-expressing cells. Bound EVs were quantified using flow cytometry. (2) We also showed that membrane-bound Gaussia luciferase allows the detection of cancer-derived EVs in blood of tumor-bearing animals. Our analytical and validation platform should be applicable to identify antigens on EVs from any tumor type.

Retrospective Evaluation of Thromboembolism Risk in Ovarian Cancer Patients Treated with Bevacizumab.

BACKGROUND: Bevacizumab is used in addition to standard, platinum-based chemotherapy to treat advanced-stage ovarian cancer patients. Thrombosis is a well-documented adverse effect of bevacizumab. OBJECTIVE: The aim of this study was to identify predictive parameters for thromboembolic events in ovarian cancer patients and to explain how bevacizumab increases the risk of these events. PATIENTS AND METHODS: Fifty-seven FIGO stage III ovarian cancer patients who underwent cytoreductive surgery and chemotherapy were identified and included in this retrospective study. Twenty-six patients were treated with carboplatin and paclitaxel (CP) only (control group), and 31 patients received CP with bevacizumab (study group). The two groups were compared with regard to thrombosis risk factors and laboratory parameters (total leukocytes, platelet count, hemoglobin, APTT, prothrombin time, INR, fibrinogen levels, D-dimer concentration) before treatment, after each course of chemotherapy, and during thromboembolic events. RESULTS: Only patients in the group receiving bevacizumab experienced venous thromboembolism (VTE) (p=0.03, χ² test). VTE occurred on average at the 13th cycle of chemotherapy. Patients who experienced VTE had increased BMI before chemotherapy as compared to patients with no thromboembolic event (27.2 vs. 23.3, p=0.005, Mann-Whitney test). D-dimer concentration before treatment was also elevated more in patients affected by VTE (3132.5) than in the non-VTE group (956.43) (p=0.0007, Mann-Whitney test). During the first four administrations of chemotherapy in patients with future VTE, there was a reduction in D-dimer concentration and an extension of APTT. A D-Dimer level higher than 485 ng/mL prior to first chemotherapy indicates for a risk of VTE with 94% sensitivity and 36% specificity. CONCLUSIONS: An elevated D-dimer level and high BMI before chemotherapy are risk factors for VTE in ovarian cancer patients receiving bevacizumab. Bevacizumab possibly increases the risk for VTE.

Paraneoplastic neurological syndromes associated with ovarian tumors.

INTRODUCTION: Paraneoplastic neurological syndromes (PNS) are neurologic deficits triggered by an underlying remote tumor. PNS can antedate clinical manifestation of ovarian malignancy and enable its diagnosis at an early stage. Interestingly, neoplasms associated with PNS are less advanced and metastasize less commonly than those without PNS. This suggests that PNS may be associated with a naturally occurring antitumor response. METHODS: We review the literature on the diagnosis, pathogenesis and management of PNS associated with ovarian tumors: paraneoplastic cerebellar degeneration (PCD) and anti-N-methyl-D-aspartate receptor (anti-NMDAR) encephalitis. An approach to the diagnostic workup of underlying tumors is discussed. RESULTS: PCD can precede the manifestation of ovarian carcinoma. Anti-NMDAR encephalitis in young women appears often as a result of ovarian teratoma. Since ovarian tumors and nervous tissue share common antigens (e.g., cdr2, NMDAR), autoimmune etiology is a probable mechanism of these neurologic disorders. The concept of cross-presentation, however, seems insufficient to explain entirely the emergence of PNS. Early resection of ovarian tumors is a significant part of PNS management and improves the outcome. CONCLUSIONS: The diagnosis of PNS potentially associated with ovarian tumor indicates a need for a thorough diagnostic procedure in search of the neoplasm. In some patients, explorative laparoscopy/laparotomy can be considered.