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.

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.

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.

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.

The nutritional supplement taurine activates p53-dependent and independent tumor suppressor mechanisms in various cellular models of ovarian cancer.

Loss of treatment-induced ovarian carcinoma (OC) growth suppression poses a major clinical challenge because it leads to disease recurrence. Therefore, there is a compelling need for well- -tolerated approaches that can support tumor growth-suppression after therapy is stopped. We have profiled ascites as OC tumor microenvironments to search for potential non-toxic soluble components that would activate tumor suppressor pathways in OC cells. Our investigations revealed that low levels of taurine, a non-proteogenic sulfonic amino acid, were present within OC ascites. Taurine supplementation, beyond levels found in ascites, induced growth suppression without causing cytotoxicity in various OC cells, including chemotherapy-resistant cell clones and patient-derived organoids representing primary or chemotherapy recovered disease. Inhibition of proliferation by taurine was linked to increased mutant or wild-type p53 proteins binding to DNA, induction of p21, and independently of p53, TIGAR expression. Taurine-induced activation of p21 and TIGAR was associated with suppression of cell-cycle progression, glycolysis, and mitochondrial respiration. Expression of p21 or TIGAR in OC cells mimicked taurine-induced growth suppression. Our studies support the potential therapeutic value of taurine supplementation in OC.

Mass spectrometry imaging in gynecological cancers: the best is yet to come.

Mass spectrometry imaging (MSI) enables obtaining multidimensional results simultaneously in a single run, including regiospecificity and m/z values corresponding with specific proteins, peptides, lipids, etc. The knowledge obtained in this way allows for a multifaceted analysis of the studied issue, e.g., the specificity of the neoplastic process and the search for new therapeutic targets. Despite the enormous possibilities, this relatively new technique in many aspects still requires the development or standardization of analytical protocols (from collecting biological material, through sample preparation, analysis, and data collection, to data processing). The introduction of standardized protocols for MSI studies, with its current potential to extend diagnostic and prognostic capabilities, can revolutionize clinical pathology. As far as identifying ovarian cancer subtypes can be challenging, especially in poorly differentiated tumors, developing MSI-based algorithms may enhance determining prognosis and tumor staging without the need for extensive surgery and optimize the choice of subsequent therapy. MSI might bring new solutions in predicting response to treatment in patients with endometrial cancer. Therefore, MSI may help to revolutionize the future of gynecological oncology in terms of diagnostics, treatment, and predicting the response to therapy. This review will encompass several aspects, e.g., contemporary discoveries in gynecological cancer research utilizing MSI, indicates current challenges, and future perspectives on MSI.

Extracellular Matrix Modulates Outgrowth Dynamics in Ovarian Cancer.

Ovarian carcinoma (OC) forms outgrowths that extend from the outer surface of an afflicted organ into the peritoneum. OC outgrowth formation is poorly understood due to the limited availability of cell culture models examining the behavior of cells that form outgrowths. Prompted by immunochemical evaluation of extracellular matrix (ECM) components in human tissues, laminin and collagen-rich ECM-reconstituted cell culture models amenable to studies of cell clusters that can form outgrowths are developed. It is demonstrated that ECM promotes outgrowth formation in fallopian tube non-ciliated epithelial cells (FNE) expressing mutant p53 and various OC cell lines. Outgrowths are initiated by cells that underwent outward translocation and retained the ability to intercalate into mesothelial cell monolayers. Electron microscopy, optical coherence tomography, and small amplitude oscillatory shear experiments reveal that increased ECM levels led to increased fibrous network thickness and high shear elasticity of the microenvironment. These physical characteristics are associated with outgrowth suppression. The low ECM microenvironment mimicks the viscoelasticity of malignant peritoneal fluid (ascites) and supports cell proliferation, cell translocation, and outgrowth formation. These results highlight the importance of the ECM microenvironment in modulating OC growth and can provide additional insights into the mode of dissemination of primary and recurrent ovarian tumors.

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.

Frequency-based dispersion Lamb-dip spectroscopy in a high finesse optical cavity.

Frequency-based cavity mode-dispersion spectroscopy (CMDS), previously applied for Doppler-limited molecular spectroscopy, is now employed for the first time for saturation spectroscopy. Comparison with two intensity-based, cavity-enhanced absorption spectroscopy techniques, i.e. cavity mode-width spectroscopy (CMWS) and the well-established cavity ring-down spectroscopy (CRDS), shows the predominance of the CMDS. The method enables measurements in broader pressure range and shows high immunity of the Lamb dip position to the incomplete model of saturated cavity mode shape. Frequencies of transitions from the second overtone of CO are determined with standard uncertainty below 500 Hz which corresponds to relative uncertainty below 3 × 10-12. The pressure shift of the Lamb dips, which has not been detected for these transitions in available literature data, is observed.

Serum visfatin as a metabolic biomarker in obese patients with gestational diabetes mellitus.

BACKGROUND: Visfatin is an adipokine produced and secreted by the adipose tissue. It exerts an insulin-like effect by the insulin receptor-1 and has a hypoglycemic effect. We aimed to investigate how serum visfatin changes in women with gestational diabetes mellitus (GDM), and whether it is predictive of neonatal outcomes. METHODS: Visfatin levels were prospectively measured in peripheral blood serum by enzyme immunoassay in 210 pregnant women, 156 of which were diagnosed with GDM, 18 of which suffered from pregnancy-induced hypertension (PIH) and 36 healthy controls. RESULTS: Patients with obesity class II (median=2.562 ng/mL) and class III (median=6.2940 ng/mL) had higher serum visfatin than overweight patients (median=0.735 ng/mL); (Mann-Whitney U test, P=0.037 and P=0.023, respectively). In GDM patients with BMI above 30, serum visfatin was associated to glycosylated hemoglobin (Spearman correlation test, R=0.26, P=0.045). Women with BMI above 25 treated with insulin had lower serum visfatin levels than those treated with diet only (Mann-Whitney U test, P=0.045). No correlation was found between visfatin and parameters of lipid profile such as HDL, LDL, or triglycerides (Spearman correlation tests, R=-0.051, -0.1, 0.0019; P=0.54, 0.29, 0.98, respectively). We observed that visfatin was not associated with birth weight (Spearman correlation test, R=-0.014, P=0.86) or adverse neonatal outcome as measured by umbilical artery pH below 7.25 (Mann-Whitney U test, P=0.55) or Apgar score below 10 (Mann-Whitney U test, P=0.21). CONCLUSIONS: In GDM patients with higher BMI, serum visfatin was elevated, correlated positively with glycosylated hemoglobin, and decreased upon treatment with insulin therapy.

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.

Ultrahigh finesse cavity-enhanced spectroscopy for accurate tests of quantum electrodynamics for molecules.

We report the most accurate, to the best of our knowledge, measurement of the position of the weak quadrupole S(2) 2-0 line in $ {{\rm D}_2} $D2. The spectra were collected with a frequency-stabilized cavity ringdown spectrometer (FS-CRDS) with an ultrahigh finesse optical cavity ($ {\cal F} = 637 000 $F=637000) and operating in the frequency-agile, rapid scanning spectroscopy (FARS) mode. Despite working in the Doppler-limited regime, we reached 40 kHz of statistical uncertainty and 161 kHz of absolute accuracy, achieving the highest accuracy for homonuclear isotopologues of molecular hydrogen. The accuracy of our measurement corresponds to the fifth significant digit of the leading term in quantum electrodynamics (QED) correction. We observe $ 2.3\sigma $2.3σ discrepancy with the recent theoretical value.

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.

High levels of AAV vector integration into CRISPR-induced DNA breaks.

Adeno-associated virus (AAV) vectors have shown promising results in preclinical models, but the genomic consequences of transduction with AAV vectors encoding CRISPR-Cas nucleases is still being examined. In this study, we observe high levels of AAV integration (up to 47%) into Cas9-induced double-strand breaks (DSBs) in therapeutically relevant genes in cultured murine neurons, mouse brain, muscle and cochlea. Genome-wide AAV mapping in mouse brain shows no overall increase of AAV integration except at the CRISPR/Cas9 target site. To allow detailed characterization of integration events we engineer a miniature AAV encoding a 465 bp lambda bacteriophage DNA (AAV-λ465), enabling sequencing of the entire integrated vector genome. The integration profile of AAV-465λ in cultured cells display both full-length and fragmented AAV genomes at Cas9 on-target sites. Our data indicate that AAV integration should be recognized as a common outcome for applications that utilize AAV for genome editing.

High-accuracy and wide dynamic range frequency-based dispersion spectroscopy in an optical cavity.

A spectroscopic method free from systematic errors is desired for many challenging applications of gas detection. Although existing cavity-enhanced techniques exhibit very high precision, their accuracy strongly depends on propagation of the light amplitude through an optical system and its detection. Here, we demonstrate that the frequency-based molecular dispersion spectroscopy, involving sub-Hz-level precision in frequency measurements of optical cavity resonances, leads to sub-per-mille accuracy and a wide dynamic range, both previously unattainable by any other spectroscopic technique. The method offers great sensitivity of 5×10-11 cm-1, high speed, limited only by the fundamental response time of the cavity, and traceability of both axes of the spectrum to the primary frequency standard. All these features are necessary for convenient realization of comprehensive molecular spectroscopy from Doppler up to collisional regime without changing the spectroscopic method and modification of the experimental setup. Moreover, the presented approach does not require linear, high-bandwidth nor phase-sensitive detectors and can be directly implemented in existing cavity-enhanced spectrometers utilizing either continuous-wave or coherent broadband radiation. We experimentally prove the predominance of frequency-based spectroscopy over intensity-based one. Our results motivate replacement of intensity-based absorption spectroscopy with a pure frequency-based dispersion one in applications where the highest accuracy is required.

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.

Allele-specific gene editing prevents deafness in a model of dominant progressive hearing loss.

Since most dominant human mutations are single nucleotide substitutions1,2, we explored gene editing strategies to disrupt dominant mutations efficiently and selectively without affecting wild-type alleles. However, single nucleotide discrimination can be difficult to achieve3 because commonly used endonucleases, such as Streptococcus pyogenes Cas9 (SpCas9), can tolerate up to seven mismatches between guide RNA (gRNA) and target DNA. Furthermore, the protospacer-adjacent motif (PAM) in some Cas9 enzymes can tolerate mismatches with the target DNA3,4. To circumvent these limitations, we screened 14 Cas9/gRNA combinations for specific and efficient disruption of a nucleotide substitution that causes the dominant progressive hearing loss, DFNA36. As a model for DFNA36, we used Beethoven mice5, which harbor a point mutation in Tmc1, a gene required for hearing that encodes a pore-forming subunit of mechanosensory transduction channels in inner-ear hair cells6. We identified a PAM variant of Staphylococcus aureus Cas9 (SaCas9-KKH) that selectively and efficiently disrupted the mutant allele, but not the wild-type Tmc1/TMC1 allele, in Beethoven mice and in a DFNA36 human cell line. Adeno-associated virus (AAV)-mediated SaCas9-KKH delivery prevented deafness in Beethoven mice up to one year post injection. Analysis of current ClinVar entries revealed that ~21% of dominant human mutations could be targeted using a similar approach.

Autoimmune response in lung cancer patients with neurological paraneoplastic syndromes.

AIM OF THE STUDY: The aim of this study was to evaluate granzyme B, perforin and FasL expression in peripheral blood mononuclear cells (PBMCs) in lung cancer patients and in paraneoplastic neurological syndromes (PNS). CLINICAL RATIONALE FOR THE STUDY: Cellular immune response is activated as part of anti-tumour reaction of the malignancy-bearing host. Paraneoplastic neurological syndromes (PNS) are defined as indirect effects of cancer on the nervous system and are considered immune-mediated. Such stimulation of the immune system may limit the aggressiveness of cancer and the development of metastasis, and thereby improve survival. Granzyme B and perforin pathway, and Fas ligand (FasL) - Fas receptor interaction play an important role in cytotoxic response. MATERIALS AND METHODS: Fifty-two patients were included in the study: 28 subjects with PNS and 24 subjects with lung cancer. PNS cases were diagnosed according to the Graus criteria. The presence of onconeural antibodies (anti-Hu/anti-Ri/anti-Yo/anti-Ma/Ta/anti-CV2/anti-amphiphysin/anti-myelin/anti-neuroendothelium/anti-MAG/anti-GAD) was detected with indirect immunofluorescence and confirmed with Line Blotting. The expression of granzyme B, perforin and FasL was detected in PBMCs with ELISA. RESULTS: PPBMC-FasL expression was increased in lung cancer compared to other patient groups. The granzyme to FasL ratio was significantly higher in lung cancer patients with peripheral than with central PNS involvement. In a multiple regression model, sex was an independent factor influencing PBMC expression of granzyme and perforin. CONCLUSIONS: FasL expression in PBMCs is up-regulated in lung cancer patients. The interplay between granzyme B and FasL may be involved in the development of PNS at the level of the peripheral and the central nervous systems in different manners. Gender is associated with PBMC expression of granzyme B and perforin in lung cancer patients. CLINICAL IMPLICATIONS: The novel findings that we report broaden the current knowledge on PNS pathomechanism, with aspects that have not been previously explored. Our findings provide a rationale for further exploration of the granzyme B/FasL pathway with regards to its potential diagnostic value. However, our study is preliminary and needs further research, especially in the context of the prognostic value of the proposed markers.

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.