Proteomic profiles of peritoneal fluid-derived small extracellular vesicles correlate with patient outcome in ovarian cancer.

Cancer-derived small extracellular vesicles (sEVs) are capable of modifying the tumor microenvironment and promoting tumor progression. Ovarian cancer (OvCa) is a lethal malignancy that preferentially spreads through the abdominal cavity. Thus, the secretion of such vesicles into the peritoneal fluid could be a determinant factor in the dissemination and behavior of this disease. We designed a prospective observational study to assess the impact of peritoneal fluid-derived sEVs (PFD-sEVs) in OvCa clinical outcome. For this purpose, 2 patient cohorts were enrolled: patients with OvCa who underwent a diagnostic or cytoreductive surgery and nononcological patients, who underwent abdominal surgery for benign gynecological conditions and acted as the control group. Systematic extraction of PFD-sEVs from surgical samples enabled us to observe significant quantitative and qualitative differences associated with cancer diagnosis, disease stage, and platinum chemosensitivity. Proteomic profiling of PFD-sEVs led to the identification of molecular pathways and proteins of interest and to the biological validation of S100A4 and STX5. In addition, unsupervised analysis of PFD-sEV proteomic profiles in high-grade serous ovarian carcinomas (HGSOCs) revealed 2 clusters with different outcomes in terms of overall survival. In conclusion, comprehensive characterization of PFD-sEV content provided a prognostic value with potential implications in HGSOC clinical management.

Reliably making the primary diagnosis of mesothelioma utilizing serous fluid cytology specimens: an institutional experience.

INTRODUCTION: The diagnosis of mesothelioma has historically been challenging, especially on serous fluid cytology (SFC). Distinguishing between reactive and neoplastic mesothelial cells can be difficult on cytomorphology alone. However, additional ancillary tests, such as BRCA1 associated protein-1 immunohistochemistry and fluorescence in situ hybridization for cyclin-dependent kinase inhibitor 2A deletion, can provide a sensitive and highly specific method of proving malignancy. MATERIALS AND METHODS: SFC specimens diagnosed as mesothelioma, suspicious for mesothelioma (SM), and atypical mesothelial cells (AMCs) since 2012 were identified by querying the laboratory information system. Clinical data and pathologic parameters were gathered. RESULTS: One hundred ten cases of mesothelioma, SM, and AMC were identified. Of these, 61 cases had a definitive diagnosis of mesothelioma on SFC. Average age at SFC diagnosis was 67 years (26-87 years), with most patients being male (67%). Out of the 61 cases, 11 cases (18%) had an initial diagnosis of mesothelioma made on SFC specimens, with 5 of these 11 cases being in patients that never received a histologic diagnosis of mesothelioma. Ancillary studies were utilized in all 11 cases. An initial diagnosis of metastatic mesothelioma was made on SFC in 9 cases (15%). For 6 of these 9 cases, the SFC diagnosis was the sole diagnosis of metastatic mesothelioma without a companion histologic diagnosis. In addition, 15 cases were diagnosed as SM, with 11 of these cases following a definitive mesothelioma diagnosis. Thirty-four cases were diagnosed as AMC, with 27 cases following a definitive mesothelioma diagnosis. CONCLUSIONS: The diagnosis of mesothelioma can be reliably made on SFC with the appropriate cytomorphology criteria and/or confirmatory ancillary testing.

Plasma and peritoneal fluid cytokine profiles in patient with Essure® implant: Towards a molecular signature?

OBJECTIVE(S): Many patients with Essure® implant may experience adverse events related to the device. Although local inflammation does not appear to be the pathophysiological mechanism underlying the symptoms, systemic inflammation could play a role. In the present study, as cytokines are involved in the inflammatory process, we proposed to investigate the profile of circulating and peritoneal cytokines. STUDY DESIGN: In this retrospective study, we evaluated the levels of cytokines in peritoneal fluid (PF) as well as in plasma sample from three different groups: Essure® group, endometriosis group (known to be associated with immune dysregulation), and control group. RESULTS: There were 60 symptomatic patients with Essure® device, 30 patients with endometriosis and a control group of 30 patients. The PF levels of Interleukin-10 (IL-10), Interleukin-6 (IL-6), and Monocyte chemoattractant protein-1 (MCP-1) were statistically higher in endometriosis group than in Essure® group and control group. The plasma level of MCP-1 was higher in Essure® group than in endometriosis group and control group. The plasma level of TNF-α was higher in Essure® group than in control group. CONCLUSIONS: The chemokine MCP-1 as well as the pro-inflammatory TNF-α, are known to be increased in patients with fibromyalgia and chronic fatigue syndrome. Since patients with Essure® may exhibit symptoms similar to fibromyalgia, MCP-1 and TNF-α may be relevant markers in symptomatic patients with Essure®. Because of the lack of longitudinal data (no evaluation of postoperative cytokine profile and no assessment of the level of clinical improvement), other studies are needed to confirm these preliminary results.

Human Amniotic MSC Response in LPS-Stimulated Ascites from Patients with Cirrhosis: FOXO1 Gene and Th17 Activation in Enhanced Antibacterial Activation.

Spontaneous bacterial peritonitis (SBP) is a severe complication in patients with decompensated liver cirrhosis and is commonly treated with broad spectrum antibiotics. However, the rise of antibiotic resistance requires alternative therapeutic strategies. As recently shown, human amnion-derived mesenchymal stem cells (hA-MSCs) are able, in vitro, to promote bacterial clearance and modulate the immune and inflammatory response in SBP. Our results highlight the upregulation of FOXO1, CXCL5, CXCL6, CCL20, and MAPK13 in hA-MSCs as well as the promotion of bacterial clearance, prompting a shift in the immune response toward a Th17 lymphocyte phenotype after 72 h treatment. In this study, we used an in vitro SBP model and employed omics techniques (next-generation sequencing) to investigate the mechanisms by which hA-MSCs modify the crosstalk between immune cells in LPS-stimulated ascitic fluid. We also validated the data obtained via qRT-PCR, cytofluorimetric analysis, and Luminex assay. These findings provide further support to the hope of using hA-MSCs for the prevention and treatment of infective diseases, such as SBP, offering a viable alternative to antibiotic therapy.

Comparison of microbial abundance and diversity in uterine and peritoneal fluid in infertile patients with or without endometriosis.

INTRODUCTION: Endometriosis (EM) is a multifactorial disease that affects 10 - 15% of women of reproductive age. Additionally, 30-50% of women with EM suffer from infertility. The mechanism of infertility caused by EM has not yet been consistently explained. In recent years, studies have shown a link between infertility associated with EM and changes in the reproductive tract microbiota. METHODS: In this study, we involved 26 EM patients (8 cases of stage I-II and 18 cases of stage III-IV) and 31 control subjects who were tubal obstruction-related infertility (TORI). The samples from peritoneal fluid (PF) and uterine fluid (UF) were collected and sequenced by 16 S rRNA amplicon. RESULTS: In the comparison of microbial diversity, we found no significant differences in the microbial diversity of PF and UF between patients with stage I-II EM and those with TORI. However, there was a significant difference in microbial diversity among patients with stage III-IV EM compared to the previous two groups. Lactobacillus decreased in PF of EM compared to the control group, while it increased in UF. In PF, the abundance of Pseudomonas, Enterococcus, Dubosiella and Klebsiella was significantly higher in patients with stage III-IV compared to TORI patients. And in UF, the main differences existed between stage I-II EM compared to the other two groups. The abundance of pontibacter, aquabacterium, Rikenellaceae and so on at the genus level was significantly enriched in the EM patients with stage I-II. In the analysis based on KEGG database, EM may affect the receptivity related pathways of the endometrium by influencing changes in the uterine microbiota. CONCLUSION: Our results indicated that as EM progresses, the microorganisms in UF and PF keep changing. These changes in the microbiota, as well as the resulting alternations in gene functional classification, may play an important role in the infertility associated with EM.

Diagnostic utility of the total nucleated cell count for differentiation of septic and sterile peritoneal effusions in dogs.

BACKGROUND: Rapid and accurate diagnosis of septic peritonitis is critical for initiating appropriate medical and surgical management. OBJECTIVES: The aim of this study was to determine the diagnostic utility of the total nucleated cell count (TNCC), absolute neutrophil count, neutrophil percentage, and total protein (TP) to distinguish septic versus non-septic peritoneal effusions in dogs. METHODS: Electronic medical records were retrospectively searched for peritoneal fluid samples from 2008 to 2018 and classified as septic or non-septic based on bacterial culture and/or cytology results. Receiver operator characteristic curves (ROCs) were used to describe the overall diagnostic utility of each test, with optimal cutpoints analyzed to dichotomize continuous variables. Positive and negative likelihood ratios were calculated at these cutpoints. RESULTS: A total of 166 unique samples, including 87 septic and 79 non-septic peritoneal effusions, were included. There were no significant differences in dog sex, age, or days hospitalized between groups. Septic effusions had significantly higher TP, TNCC, absolute neutrophil count, and neutrophil percentage compared with non-septic effusions. The area under the curve of the ROC curves was TNCC (0.80), absolute neutrophil count (0.80), neutrophil percentage (0.64), and TP (0.63). For TNCC and absolute neutrophil count, optimal cutoffs were 17.13 × 103 cells/µL and 19.88 × 103 cells/µL, resulting in positive and negative likelihood ratios of 2.39 and 0.28 and 2.85 and 0.28, respectively. CONCLUSIONS: Total nucleated cell counts and absolute neutrophil counts aid in the differentiation of septic and non-septic peritoneal effusions with similar diagnostic utility but are not sufficiently sensitive or specific to use without concurrent microscopic evaluation.

Viscosity and Characteristics of Malignant Ascites.

BACKGROUND/AIM: Malignant ascites is a common condition in patients with terminal cancer. Treatments, such as diuretics, percutaneous drainage of ascites, and abdominal vein shunting have been advocated. However, these treatments have not achieved sufficient palliative effects. Therefore, the development of innovative therapies is mandated, especially for new therapies that require the creation of a fluid simulation of malignant ascites. However, there have been no previous studies on the physical properties of malignant ascites, including viscosity, which are necessary for the development of such a fluid. Therefore, we prospectively investigated the physical properties of malignant ascites. PATIENTS AND METHODS: This single-center, prospective, observational study included 30 patients between November 2021 and January 2023. The primary endpoint was the viscosity of the malignant ascites, and the secondary endpoints included other viscosity studies, biochemical tests, and the presence of malignant cells in the ascites. RESULTS: The median viscosity was 1.105 mPa*S. The viscosity of malignant ascites tended to decrease with increasing temperature, which is common for liquids. Malignant ascites fluid containing malignant cells tended to be more viscous than ascites fluid without malignant cells; furthermore, albumin levels tended to be higher in the former than in the latter. CONCLUSION: Malignant ascites' median viscosity was 1.105 mPa*S. Correlation between viscosity and temperature showed a decreasing trend. These findings contribute valuable insights for future malignant ascites management and device development.

Cascaded elasto-inertial separation of malignant tumor cells from untreated malignant pleural and peritoneal effusions.

Separation of malignant tumor cells (MTCs) from large background cells in untreated malignant pleural and peritoneal effusions (MPPEs) is critical for improving the sensitivity and efficiency of cytological diagnosis. Herein, we proposed a cascaded elasto-inertial cell separation (CEICS) device integrating an interfacial elasto-inertial microfluidic channel with a symmetric contraction expansion array (CEA) channel for pretreatment-free, high-recovery-ratio, and high-purity separation of MTCs from clinical MPPEs. First, the effects of flow-rate ratio, cell concentration, and cell size on separation performances in two single-stage channels were investigated. Then, the performances of the integrated CEICS device were characterized using blood cells spiked with three different tumor cells (MCF-7, MDA-MB-231, and A549 cells) at a high total throughput of 240 µL min-1. An average recovery ratio of ∼95% and an average purity of ∼61% for the three tumor cells were achieved. Finally, we successfully applied the CEICS device for the pretreatment-free separation of MTCs from clinical MPPEs of different cancers. Our CEICS device may provide a preparation tool for improving the sensitivity and efficiency of cytological examination.

Overexpression of Fut 2, 4, and 8, and nuclear localization of Fut 4 in ovarian cancer cell lines induced by ascitic fluids from epithelial ovarian cancer patients.

Fucosyltransferases (Fut) regulate the fucosylation process associated with tumorogenesis in different cancer types. Ascitic fluid (AF) from patients diagnosed with advanced stage of epithelial ovarian cancer (EOC) is considered as a dynamic tumor microenvironment associated with poor prognosis. Previous studies from our laboratory showed increased fucosylation in SKOV-3 and OVCAR-3, cancer-derived cell lines, when these cells were incubated with AFs derived from patients diagnosed with EOC. In the present work we studied three fucosyltransferases (Fut 2, Fut 4, and Fut 8) in SKOV-3, OVCAR-3 and CAOV-3 cell lines in combination with five different AFs from patients diagnosed with this disease, confirming that all tested AFs increased fucosylation. Then, we demonstrate that mRNAs of these three enzymes were overexpressed in the three cell lines under treatment with AFs. SKOV-3 showed the higher overexpression of Fut 2, Fut 4, and Fut 8 in comparison with the control condition. We further confirmed, in the SKOV-3 cell line, by endpoint PCR, WB, and confocal microscopy, that the three enzymes were overexpressed, being Fut 4 the most overexpressed enzyme compared to Fut 2 and Fut 8. These enzymes were concentrated in vesicular structures with a homogeneous distribution pattern throughout the cytoplasm. Moreover, we found that among the three enzymes, only Fut 4 was located inside the nuclei. The nuclear location of Fut 4 was confirmed for the three cell lines. These results allow to propose Fut 2, Fut 4, and Fut 8 as potential targets for EOC treatment or as diagnostic tools for this disease.

Ascites fluid calprotectin level is highly accurate in diagnosing spontaneous bacterial peritonitis: a preliminary proof of concept prospective study.

Ascites is the most common complication of liver cirrhosis. Spontaneous bacterial peritonitis (SBP) is a common complication of ascites. The diagnosis is made by an ascitic fluid polymorphonuclear (PMN) cell count of ≥ 250/mm3. However, no other diagnostic test is present for the diagnosis of SBP. The aim of the study present study is to assess the diagnostic yield of ascitic calprotectin in SBP, and to explore whether it can predict disease stage. We performed a single center proof-of-concept prospective study including all patients with cirrhosis and ascites who underwent paracentesis. Overall, 31 patients were included in the study. Eight patients had SBP vs. 23 patients without SBP. Ascitic calprotectin level was 77.4 ± 86.5 µg/mL in the SBP group, as compared to 16.1 ± 5.6 µg/mL in the non-SBP group (P = 0.001). An ascitic calprotectin cut-off value of > 21 µg/mL was associated with sensitivity and specificity of 85.7% and 89.5%, respectively, with ROC of 0.947 (95% CI 0.783 to 0.997, P < 0.0001). Notably, ascitic calprotectin did not had a prognostic value in cirrhosis stage and prognosis. Ascitic calprotectin was highly accurate in the diagnosis of SBP. It can be a serve as adjunct for indefinite cases of SBP.

Use of multiple admission variables better predicts intestinal strangulation in horses with colic than peritoneal or the ratio of peritoneal:blood l-lactate concentration.

BACKGROUND: Early identification of strangulating obstruction (SO) in horses with colic improves outcomes, yet early diagnosis of horses requiring surgery for SO often remains challenging. OBJECTIVES: To compare blood and peritoneal fluid l-lactate concentrations, peritoneal:blood l-lactate ratio, peritoneal minus blood (peritoneal-blood) l-lactate concentration and other clinical variables for predicting SO and SO in horses with small intestinal lesions (SO-SI) and then to develop a multivariable model to predict SO and SO-SI. STUDY DESIGN: Retrospective cohort. METHODS: A total of 197 equids admitted to a referral institution for colic between 2016 and 2019 that had peritoneal fluid analysis performed at admission were included. Twenty-three admission variables were evaluated individually for the prediction of a SO or SO-SI and then using multivariable logistic regression. Odds ratios (ORs) with 95% confidence intervals (CI) and area under the curve of the receiver operator characteristic (AUC ROC) were calculated. RESULTS: All variables performed better in the model than individually. The final multivariable model for predicting SO included marked abdominal pain (OR 5.31, CI 1.40-20.18), rectal temperature (OR 0.30, CI 0.14-0.64), serosanguineous peritoneal fluid (OR 35.34, CI 10.10-122.94), peritoneal-blood l-lactate (OR 1.77, CI 1.25-2.51), and peritoneal:blood l-lactate ratio (OR 0.36, CI 0.18-0.72). The AUC ROC was 0.91. The final multivariable model for predicting SO-SI included reflux volume (OR 0.69, CI 0.56-0.86), blood l-lactate concentration (OR 0.43, CI 0.22-0.87), serosanguineous peritoneal fluid (OR 4.99, CI 1.26-19.74), and peritoneal l-lactate concentration (OR 3.77, CI 1.82-7.81). MAIN LIMITATIONS: Retrospective, single-hospital study design. CONCLUSIONS: Blood and peritoneal fluid l-lactate concentrations should be interpreted in conjunction with other clinical variables. The relationship between peritoneal and blood l-lactate concentration for predicting SO or SO-SI was complex when included in a multivariable model. Models to predict SO probably vary based on lesion location.

Diagnosis of peritoneal metastasis in an unruptured hepatocellular carcinoma on ascitic fluid cytology: A rare scenario with brief review of literature.

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy in adults occurring in a background of cirrhosis. Peritoneal dissemination of HCC is an unusual presentation with an incidence of 2%-16%. Peritoneal metastasis of an unruptured HCC is extremely uncommon. Despite low yield, ascitic fluid cytology serves as a valuable tool for diagnostic evaluation in a patient of cirrhosis with suspicion of malignant transformation. We present a rare case scenario in an elderly female with cirrhosis where the diagnosis of peritoneal metastasis was established on ascitic fluid cytology and confirmed by immunocytochemistry. This report illustrates the unique clinical presentation of an unruptured HCC with its cytological features and a brief review of literature.

Ovarian cancer relies on the PDGFRß-fibronectin axis for tumorsphere formation and metastatic spread.

High-grade serous ovarian cancer (HGSOC) is the deadliest gynecological malignancy. The most common form of metastatic spread of HGSOC is transcoelomic dissemination. In this process, detached cells from the primary tumor aggregate as tumorspheres and promote the accumulation of peritoneal ascites. This represents an early event in HGSOC development and is indicative of poor prognosis. In this study, based on tumorspheres isolated from ascitic liquid samples from HGSOC patients, ovarian cancer spheroid 3D cultures, and in vivo models, we describe a key signal for tumorsphere formation in HGSOC. We report that platelet-derived growth factor receptor beta (PDGFRß) is essential for fibronectin-mediated cell clustering of ovarian cancer cells into tumorspheres. This effect is mediated by the kinase NUAK family SNF1-like kinase 1 (NUAK1) and blocked by PDGFRß pharmacological or genetic inhibition. In the absence of PDGFRß, ovarian cancer cells can be provided with fibronectin by cancer-associated fibroblasts to generate chimeric spheroids. This work provides new insights that uncover potential targets to prevent peritoneal dissemination, the main cause of advanced disease in HGSOC patients.

Flow cytometry in the detection of circulating tumor cells in neoplastic effusions.

PURPOSE: Despite its limitations, the cytology of body fluids is widely used in diagnosing neoplastic cells. Flow cytometry detects and identifies individual cells, enabling the detection of circulating tumor cells and facilitating diagnosis. This study compared the diagnostic utility of flow cytometry and cytology for detecting cancer cells in peritoneal and pleural fluids. METHODOLOGY: We used flow cytometry and cytology to examine 119 pleural and peritoneal effusions received for routine screening. Antibodies against clusters of differentiation 45 (CD45), 14 (CD14), and Epithelial cell adhesion molecule (EpCAM) were used to detect malignant cells. Based on combined clinical and diagnostic information, 37 fluid specimens were malignant, and 77 were benign. RESULTS: Flow cytometry correctly identified 34 cancers, while cytology identified 26 cancers (sensitivity 91.89 % vs. 70.27, respectively). Both methods had equal specificity (98.7 %). At a cut-off of > 0.29 % EpCAM(+) cells to all cells in the samples, flow cytometry accurately detected cancer cells, achieving 89.2 % sensitivity, 90.9 % specificity, and an AUC of 0.959 (p < 0.001). CONCLUSION: Flow cytometry improves the detection of epithelial cancer cells in peritoneal and pleural fluids compared to conventional cytology. Due to similar specificity and higher sensitivity, flow cytometry offers a promising alternative to cytology for patient screening.

Characterizing the Immune Environment in Peritoneal Carcinomatosis: Insights for Novel Immunotherapy Strategies.

BACKGROUND OR PURPOSE: Carcinomatosis, a distinct pattern of metastatic cancer in the peritoneal cavity, poses challenges for treatment and has limited therapeutic options. Understanding the immune environment of peritoneal surface malignancies is crucial for developing effective immunotherapeutic approaches. This study characterizes soluble immune mediators in the peritoneal fluid of patients with and without carcinomatosis to identify targets for novel treatment strategies. PATIENTS AND METHODS: Serum and peritoneal fluid samples were collected from surgical patients, and a multianalyte analysis was performed using the Luminex platform. Patient characteristics, tumor sites, and sample collection details were recorded. Soluble immune mediator levels were measured and compared between peritoneal fluid and serum samples and among clinical subgroups. Statistical analysis was conducted to assess differences in analyte concentrations and correlations between samples. RESULTS: There were 39 patients included in the study, with varying surgical indications. Significant differences were observed in soluble immune mediator levels between peritoneal fluid and serum, with peritoneal fluid exhibiting lower concentrations. Carcinomatosis was associated with elevated levels of proinflammatory mediators, including IL-6 and IL-8, while adaptive immune response markers were low in peritoneal fluid. CONCLUSIONS: The peritoneal immune microenvironment in carcinomatosis favors innate immunity, presenting a challenging environment for effective antitumor response. High levels of proinflammatory mediators suggest potential targets for intervention, such as the IL-6 axis, FGF2, IL-8, and CCL2; these could be explored as potential mitigators of malignant ascites and enhance anti-tumor immune responses. These findings provide valuable insights for developing immunotherapy strategies and improving outcomes in patients with peritoneal carcinomatosis.

The diagnostic accuracy and prognostic implication of pelvic and peritoneal fluid cytology specimens in ovarian clear cell carcinoma.

INTRODUCTION: Ovarian clear cell carcinoma (OCCC) is a rare subtype of ovarian epithelial carcinoma. Patients with low-stage disease have an excellent prognosis, while the prognosis for those with high-stage disease is poor. Neoplastic cells in abdominopelvic washings upstages the patient to at least FIGO 1C3. Positive cytology confers a worse prognosis when compared to similar stage patients with negative cytology. This study aims to investigate the diagnostic performance of abdominopelvic fluid cytology specimens in cases with pure OCCC and reaffirm the importance of accurate cytologic detection and its impact on patient prognosis. MATERIALS AND METHODS: The laboratory information system was queried to identify all patients treated for ovarian clear cell carcinoma at our institution over a period of 20 years with a companion abdominopelvic fluid cytology specimen at the time of surgical resection. Cases were sorted by the FIGO stage of the corresponding oophorectomy specimen. Cytology results, patient demographics, fluid volume, immunohistochemical results, and follow-up data were recorded. RESULTS: A total of 143 cases were identified. The overall detection rate was 38%, with 54 of 143 cases positive for malignancy. Cytologic detection rates increased as FIGO stages increased. Fifty percent of stage 1C cases were upstaged on cytology alone. Ascites fluids performed better among stage 1 cases compared to pelvic wash specimens (77% detection rate versus 23%). Stage 1 patients with positive cytology trended towards a worse prognosis compared to those with negative cytology. CONCLUSIONS: Positive cytology in low stage cases of OCCC has significant prognostic and therapeutic implications. Our large cohort further underscores the importance of accurate cytologic detection and subsequent staging in this setting.