Azoospermia: Is it worth waiting for the confirmation of the semen abnormality to start an infertility assessment?

Azoospermia is found in about 1% of men in the general population and in about 10%-15% of infertile men. Upon discovery of semen analysis abnormality, another test must be performed after an interval of 3 months before any other infertility work-up. This research aimed at evaluating the benefit of waiting for the control test. This retrospective monocentric descriptive study was carried out in the fertility center of the University Hospital of Saint Etienne. All consecutive azoospermic patients diagnosed between January, 2012 and December, 2019 were included. For each patient, two consecutive semen analyses performed 3 months apart were studied. The main focas was on patients whose second semen analysis would have modified the infertility work-up. Amongst the 172 cases under study, the second semen analysis revealed the presence of sperm for three men. Only one of these 3 modified semen analyses was normal. In the observed azoospermic population, sperm was found on the second test in 1.7%. An infertility assessment is necessary after the discovery of azoospermia in the first semen analysis in 99.5%. These results suggest that it is useless to wait three stressful months before starting an infertility assessment for azoospermic population.

Impact of Nanoparticles on Male Fertility: What Do We Really Know? A Systematic Review.

The real impact of nanoparticles on male fertility is evaluated after a careful analysis of the available literature. The first part reviews animal models to understand the testicular biodistribution and biopersistence of nanoparticles, while the second part evaluates their in vitro and in vivo biotoxicity. Our main findings suggest that nanoparticles are generally able to reach the testicle in small quantities where they persist for several months, regardless of the route of exposure. However, there is not enough evidence that they can cross the blood-testis barrier. Of note, the majority of nanoparticles have low direct toxicity to the testis, but there are indications that some might act as endocrine disruptors. Overall, the impact on spermatogenesis in adults is generally weak and reversible, but exceptions exist and merit increased attention. Finally, we comment on several methodological or analytical biases which have led some studies to exaggerate the reprotoxicity of nanoparticles. In the future, rigorous clinical studies in tandem with mechanistic studies are needed to elucidate the real risk posed by nanoparticles on male fertility.

Multiparametric investigation of non functionalized-AGuIX nanoparticles in 3D human airway epithelium models demonstrates preferential targeting of tumor cells.

Liquid deposit mimicking surface aerosolization in the airway is a promising strategy for targeting bronchopulmonary tumors with reduced doses of nanoparticle (NPs). In mimicking and studying such delivery approaches, the use of human in vitro 3D culture models can bridge the gap between 2D cell culture and small animal investigations. Here, we exposed airway epithelia to liquid-apical gadolinium-based AGuIX® NPs in order to determine their safety profile. We used a multiparametric methodology to investigate the NP's distribution over time in both healthy and tumor-bearing 3D models. AGuIX® NPs were able to target tumor cells in the absence of specific surface functionalization, without evidence of toxicity. Finally, we validated the therapeutic potential of this hybrid theranostic AGuIX® NPs upon radiation exposure in this model. In conclusion, 3D cell cultures can efficiently mimic the normal and tumor-bearing airway epitheliums, providing an ethical and accessible model for the investigation of nebulized NPs.

Ex vivo detection and quantification of gold nanoparticles in human seminal and follicular fluids.

Increasing consumption of engineered nanoparticles and occupational exposure to novel, ultrafine airborne particles during the last decades has coincided with deterioration of sperm parameters and delayed fecundity. In order to prevent possible adverse health effects and ensure a sustainable growth for the nanoparticle industry, the ability to investigate the nanosized, mineralogical load of human reproductive systems is becoming a real clinical need. Toward this goal, the current study proposes two methods for the detection and quantification of engineered nanoparticles in human follicular and seminal fluid, developed with the use of well-defined 60 nm Au particles. Despite the complexity of these biological fluids, simple physical and chemical treatments allow for the precise quantification of more than 50 and 70% wt of the spiked Au nanoparticles at low µg ml-1 levels in follicular and seminal fluids, respectively. The use of electron microscopy for the detailed observation of the detected analytes is also enabled. The proposed method is applied on a small patient cohort in order to demonstrate its clinical applicability by exploring the differences in the metal and particulate content between patients with normal and low sperm count.

EGFR, KRAS, BRAF and HER2 testing in metastatic lung adenocarcinoma: Value of testing on samples with poor specimen adequacy and analysis of discrepancies.

Molecular testing on metastatic lung adenocarcinoma or on non-small cell non-squamous lung carcinoma often relies on small specimen. In this group of patient with poor specimen adequacy, we analyzed the rate of EGFR, KRAS, BRAF and HER2 mutations compared to their rate in optimal specimen. We analyzed discrepancies in molecular testing results in patients with iterative analysis on several samples. We performed a retrospective study of 1538 samples consecutively analyzed. 263/665 (39,5%) biopsies and 37/708 (5,2%) surgical specimens were considered as samples with poor specimen adequacy (p<0,0001). A lower tumor cell content was associated with a lower rate of KRAS mutation: 15,8% in samples with <10% of tumor cells or <100 tumor cells versus 29,8% in samples with >10% tumor cell and >100 tumor cells (p=0,001). KRAS mutational rate was at 11,1% in cytology specimens, significantly lower than in biopsy or surgical specimens respectively at 28,2% and 28,5% (p=0,0002). Tumor cell content was not associated with mutational rate for EGFR, BRAF and HER2 mutations. DNA quantity was not associated with mutational rate for EGFR, KRAS, BRAF and HER2. A discrepancy in molecular testing was found in 16 patients. For 5 patients there was also a discrepancy for TTF-1 expression. On the 11 without TTF-1 discrepancy, specimen adequacy was not fulfilled in 10 cases at least for tumor content. Discrepancies were found in the case of low cellularity, poor cell content or testing on cytological specimens. Tumor cell content is a crucial parameter for molecular analysis rather than the type of specimen or the DNA quantity. Discrepancies in molecular testing results are rare but might suggest the presence of another tumor type, the emergence of another clone or a molecular testing in a sample with low cell content.

CA9 as a biomarker in preoperative biopsy of small solid renal masses for diagnosis of clear cell renal cell carcinoma.

PURPOSE: The purpose of our study was to test the utility of CA9 expression in preoperative biopsy samples to identify the ccRCC. MATERIALS AND METHODS: A total of 55 patients with a small solid renal mass (≤4 cm) entered into this study. The immunohistochemical staining (51 samples) and RT-PCR (33 samples) were performed to detect CA9 expression. RESULTS: For immunohistochemistry detection, CA9 was positive in 31/34 of biopsy samples of ccRCCs. CA9 was also positive in five suspected diagnosis of ccRCC. For RT-PCR detection, CA9 was positive in 25/25 biopsy samples of ccRCCs. The diagnostic accuracy of CA9 expression for ccRCC was 100%. RT-PCR was performed in four biopsies where immunohistochemistry could not be performed because of limited tissue materials or necrosis. Two ccRCCs with a negative staining by immunohistochemistry were CA9 positive by RT-PCR. CONCLUSIONS: CA9 may be potentially useful biomarker in help making a diagnosis of ccRCC in the biopsy of renal mass. RT-PCR might be a preferred method to immunohistochemistry for the detection of CA9 in renal biopsy samples.

EBUS-TBNA Can Distinguish Sarcoid-Like Side Effect of Nivolumab Treatment from Tumor Progression in Non-Small Cell Lung Cancer.

With the expansion of immunotherapy in the treatment of lung cancer, clinicians have to face new clinical pictures and adapt their practice. We report the case of a 69-year-old man diagnosed with non-small cell lung cancer using endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) and treated with nivolumab as second-line therapy. After 8 injections of nivolumab, a new CT and PET scan revealed massive growth and increase in metabolism of hilar and mediastinal lymph nodes, whereas the size and metabolism of the left upper lobe lesion were reduced. A new EBUS-TBNA was thus performed and showed an epithelioid cell reaction compatible with sarcoidosis in the 3 punctured lymph nodes (stations 4R, 11L, 7). In the absence of cancer evolution, nivolumab was continued, and the CT after the twelfth injection showed stability.

Affinity of Indocyanine Green in the Detection of Colorectal Peritoneal Carcinomatosis.

Indocyanine green (ICG) is increasingly being used in digestive oncology. In colorectal cancer, ICG can be used to detect lymph node metastasis and hepatic metastasis on the surface of the liver. In peritoneal carcinomatosis, it was previously suspected that the diffusion of ICG in the tumor mass was due to the enhanced permeability and retention effect; however, this phenomenon has not been clearly demonstrated. Using bevacizumab, an antibody directed against vascular endothelial growth factor that consequently inhibits neoangiogenesis, we sought to confirm the mode of ICG diffusion. We compared the fluorescence of peritoneal carcinomatosis nodules from patients who had previously received bevacizumab during their oncologic treatment with those who did not receive this therapy. The sensitivity of the carcinomatosis nodule fluorescence was higher in the patients who did not receive bevacizumab compared with those who received the drug (76.3% and 65.0%, respectively). The rate of false-negative results was higher in the bevacizumab group than in the group that did not receive the drug (53.8% and 42.9%, respectively). Using bevacizumab, we demonstrate that the enhanced permeability and retention effect causes ICG accumulation in peritoneal carcinomatosis resulting from colorectal cancer.

Micron-sized and submicron-sized aerosol deposition in a new ex vivo preclinical model.

BACKGROUND: The knowledge of where particles deposit in the respiratory tract is crucial for understanding the health effects associated with inhaled drug particles. METHOD: An ex vivo study was conducted to assess regional deposition patterns (thoracic vs. extrathoracic) of radioactive polydisperse aerosols with different size ranges [0.15 µm-0.5 µm], [0.25 µm-1 µm] and [1 µm-9 µm]. SPECT/CT analyses were performed complementary in order to assess more precisely the regional deposition of aerosols within the pulmonary tract. Experiments were set using an original respiratory tract model composed of a human plastinated head connected to an ex vivo porcine pulmonary tract. The model was ventilated by passive expansion, simulating pleural depressions. Aerosol was administered during nasal breathing. RESULTS: Planar scintigraphies allowed to calculate the deposited aerosol fractions for particles in the three size ranges from sub-micron to micron The deposited fractions obtained, for thoracic vs. extra-thoracic regions respectively, were 89 ± 4 % vs. 11 ± 4 % for [0.15 µm-0.5 µm], 78 ± 5 % vs. 22 ± 5 % for [0.25 µm-1 µm] and 35 ± 11 % vs.65 ± 11 % for [1 µm-9 µm]. CONCLUSION: Results obtained with this new ex vivo respiratory tract model are in good agreement with the in vivo data obtained in studies with baboons and humans.

Improving Surgical Resection of Metastatic Liver Tumors With Near-Infrared Optical-Guided Fluorescence Imaging.

Objective The aim of this study was to investigate the feasibility and future clinical applications of near-infrared (NIR) fluorescence imaging to guide liver resection surgery for metastatic cancer to improve resection margins. Summary Background Data A subset of patients with metastatic hepatic tumors can be cured by surgery. The degree of long-term and disease-free survival is related to the quality of surgery, with the best resection defined as "R0" (complete removal of all tumor cells, as evidenced by microscopic examination of the margins). Although intraoperative ultrasonography can evaluate the surgical margins, surgeons need a new tool to perfect the surgical outcome. Methods A preliminary study was performed on 3 patients. We used NIR imaging postoperatively "ex vivo" on the resected liver tissue. The liver tumors were preoperatively labelled by intravenously injecting the patient with indocyanine green (ICG), a NIR fluorescent agent (24 hours before surgery, 0.25 mg/kg). Fluorescent images were obtained using a miniaturized fluorescence imaging system (FluoStic, Fluoptics, Grenoble, France). Results After liver resection, the surgical specimens from each patient were sliced into 10-mm sections in the operating room and analyzed with the FluoStic. All metastatic tumors presented rim-type fluorescence. Two specimens had incomplete rim fluorescence. The pathologist confirmed the presence of R1 margins (microscopic residual resection), even though the ultrasonographic analysis indicated that the result was R0. Conclusions Surgical liver resection guided by NIR fluorescence can help detect potentially uncertain anatomical areas that may be missed by preoperative imaging and by ultrasonography during surgery. These preliminary results will need to be confirmed in a larger prospective patient series.

Cell-free RNA content in urine as a possible molecular diagnostic tool for clear cell renal cell carcinoma.

There is limited research on cell-free RNA (cf-RNA) in the urine of cancer patients. The present study was performed to detect the cf-RNA in the urine of patients with clear cell renal cell carcinoma (ccRCC). Ninety-five urine samples from ccRCC patients and 50 urine samples from control subjects were analyzed. The cf-RNA integrity index was calculated by using quantitative real-time RT-PCR assays of the small-sized fragment (106 bp) and the big-sized fragment (416 bp) in GAPDH mRNA. The initial analysis showed that cf-RNA was stable and detectable in the urine. The mean cf-RNA integrity index was significantly lower in the urine of ccRCC patients (mean: 0.07, 95%CI: 0.05-0.10) when compared with the urine from control subjects (mean: 0.25, 95%CI: 0.16-0.33) (p < 0.001). The value of the area under the receiver-operating characteristic curve by using the cf-RNA integrity index for the diagnosis of ccRCC was 0.858 with a sensitivity of 68.0% and a specificity of 92.6%. Moreover, the small-sized VEGF mRNA fragment (98 bp) was detected in 31 of 50 urine samples of patients with ccRCC and in only 2 of 50 urine samples of control subjects (p < 0.001) while the detection of the big-sized (420 bp) VEGF mRNA fragment was an infrequent event. Our findings suggest that the small-sized cf-RNA in urine was more abundant in cancer patients. The tumor-related gene VEGF mRNA fragment was detectable in the urine of cancer patients. Our finding may provide a new molecular assay for the diagnosis of renal cell carcinoma.

Testicular biodistribution of silica-gold nanoparticles after intramuscular injection in mice.

With the continuing development of nanomaterials, the assessment of their potential impact on human health, and especially human reproductive toxicity, is a major issue. The testicular biodistribution of nanoparticles remains poorly studied. This study investigated whether gold-silica nanoparticles could be detected in mouse testes after intramuscular injection, with a particular focus on their ability to cross the blood-testis barrier. To that purpose, well-characterized 70-nm gold core-silica shell nanoparticles were used to ensure sensitive detection using high-resolution techniques. Testes were collected at different time points corresponding to spermatogenesis stages in mice. Transmission electron microscopy and confocal microscopy were used for nanoparticle detection, and nanoparticle quantification was performed by atomic emission spectroscopy. All these techniques showed that no particles were able to reach the testes. Results accorded with the normal histological appearance of testes even at 45 days post sacrifice. High-resolution techniques did not detect 70-nm silica-gold nanoparticles in mouse testes after intramuscular injection. These results are reassuring about the safety of nanoparticles with regard to male human reproduction, especially in the context of nanomedicine.

Quantification of nanoparticle endocytosis based on double fluorescent pH-sensitive nanoparticles.

Amorphous silica is a particularly interesting material because of its inertness and chemical stability. Silica nanoparticles have been recently developed for biomedical purposes but their innocuousness must be carefully investigated before clinical use. The relationship between nanoparticles physicochemical features, their uptake by cells and their biological activity represents a crucial issue, especially for the development of nanomedicine. This work aimed at adapting a method for the quantification of nanoparticle endocytosis based on pH-sensitive and double fluorescent particles. For that purpose, silica nanoparticles containing two fluorophores: FITC and pHrodo(TM) were developed, their respective fluorescence emission depends on the external pH. Indeed, FITC emits a green fluorescence at physiological pH and pHrodo(TM) emits a red fluorescence which intensity increased with acidification. Therefore, nanoparticles remained outside the cells could be clearly distinguished from nanoparticles uptaken by cells as these latter could be spotted inside cellular acidic compartments (such as phagolysosomes, micropinosomes…). Using this model, the endocytosis of 60 nm nanoparticles incubated with the RAW 264.7 macrophages was quantified using time-lapse microscopy and compared to that of 130 nm submicronic particles. The amount of internalized particles was also evaluated by fluorimetry. The biological impact of the particles was also investigated in terms of cytotoxicity, pro-inflammatory response and oxidative stress. Results clearly demonstrated that nanoparticles were more uptaken and more reactive than submicronic particles. Moreover, we validated a method of endocytosis quantification.

Adsorption of lactate dehydrogenase enzyme on carbon nanotubes: how to get accurate results for the cytotoxicity of these nanomaterials.

Carbon nanotube (CNT) cytotoxicity is frequently investigated using in vitro classical toxicology assays. However, these cellular tests, usually based on the use of colorimetric or fluorimetric dyes, were designed for chemicals and may not be suitable for nanosized materials. Indeed, because of their unique physicochemical properties CNT can interfere with the assays and bias the results. To get accurate data and draw reliable conclusions, these artifacts should be carefully taken into account. The aim of this study was to evaluate qualitatively and quantitatively the interferences occurring between CNT and the commonly used lactate dehydrogenase (LDH) assay. Experiments under cell-free conditions were performed, and it was clearly demonstrated that artifacts occurred. They were due to the intrinsic absorbance of CNT on one hand and the adsorption of LDH at the CNT surface on the other hand. The adsorption of LDH on CNT was modeled and was found to fit the Langmuir model. The K(ads) and n(eq) constants were defined, allowing the correction of results obtained from cellular experiments to get more accurate data and lead to proper conclusions on the cytotoxicity of CNT.

Impact of airborne particle size, acoustic airflow and breathing pattern on delivery of nebulized antibiotic into the maxillary sinuses using a realistic human nasal replica.

PURPOSE: Improvement of clinical outcome in patients with sinuses disorders involves targeting delivery of nebulized drug into the maxillary sinuses. We investigated the impact of nebulization conditions (with and without 100 Hz acoustic airflow), particle size (9.9 µm, 2.8 µm, 550 nm and 230 nm) and breathing pattern (nasal vs. no nasal breathing) on enhancement of aerosol delivery into the sinuses using a realistic nasal replica developed by our team. METHODS: After segmentation of the airways by means of high-resolution computed tomography scans, a well-characterized nasal replica was created using a rapid prototyping technology. A total of 168 intrasinus aerosol depositions were performed with changes of aerosol particle size and breathing patterns under different nebulization conditions using gentamicin as a marker. RESULTS: The results demonstrate that the fraction of aerosol deposited in the maxillary sinuses is enhanced by use of submicrometric aerosols, e.g. 8.155 ± 1.476 mg/L of gentamicin in the left maxillary sinus for the 2.8 µm particles vs. 2.056 ± 0.0474 for the 550 nm particles. Utilization of 100-Hz acoustic airflow nebulization also produced a 2- to 3-fold increase in drug deposition in the maxillary sinuses (e.g. 8.155 ± 1.476 vs. 3.990 ± 1.690 for the 2.8 µm particles). CONCLUSIONS: Our study clearly shows that optimum deposition was achieved using submicrometric particles and 100-Hz acoustic airflow nebulization with no nasal breathing. It is hoped that our new respiratory nasal replica will greatly facilitate the development of more effective delivery systems in the future.

Toxicity of boehmite nanoparticles: impact of the ultrafine fraction and of the agglomerates size on cytotoxicity and pro-inflammatory response.

Boehmite (γ-AlOOH) nanoparticles (NPs) are used in a wide range of industrial applications. However, little is known about their potential toxicity. This study aimed at a better understanding of the relationship between the physico-chemical properties of these NPs and their in vitro biological activity. After an extensive physico-chemical characterization, the cytotoxicity, pro-inflammatory response and oxidative stress induced by a bulk industrial powder and its ultrafine fraction were assessed using RAW264.7 macrophages. Although the bulk powder did not trigger a significant biological activity, pro-inflammatory response was highly enhanced with the ultrafine fraction. This observation was confirmed with boehmite NPs synthesized at the laboratory scale, with well-defined and tightly controlled physico-chemical features: toxicity was increased when NPs were dispersed. In conclusion, the agglomerates size of boehmite NPs has a major impact on their toxicity, highlighting the need to study not only raw industrial powders containing NPs but also the ultrafine fractions representative of respirable particles.

Tumor-Derived Exosomal RNA From Fine-Needle Aspiration Supernatant as a Novel Liquid Biopsy for Molecular Diagnosis of Cancer.

Background: We hypothesized that the fine needle aspiration (FNA) supernatant from tumor might contain tumor-derived exosomes. The objective of this pilot study was to test if tumor-derived exosomal RNA could be found in FNA supernatants for molecular diagnosis of cancer. Methods: 10 FNA samples from pancreatic tumor were included. After the routine recuperation of cellular material by centrifugation, the cell-free Cytolyt liquid was collected instead of being discarded. 10 ml Cytolyt was used to isolate the exosomes. Transmission electronic microscopy (TEM) was used to examine the presence of exosomes. The exosomal marker CD63 was analyzed by flow cytometry. The exosomal RNA was extracted. RT-qPCR was performed to detect the GAPDH and the tumor marker of glypican 1 gene expression. Results: TEM confirmed the presence of exosomes from FNA supernatants. Flow cytometry showed a strong positive expression of exosome marker CD63. The concentration of exosomal RNA ranged from 18.81 to 354.75 ng/µl with an average of 81.76 ng/µl. The average exosomal RNA quantity was 1390.01 ng (range from 319.77 to 6030.75 ng) with an average 260/280 ratio of 2.12. GAPDH was detectable in all samples. Exosomal glypican 1 was detected in all samples of pancreatic ductal adenorcarcinomas (3/3) and absent from benign cystic samples (3/3). Furthermore, exosomal glypican 1 was positive in one sample with a non-contributive cytology and in one sample in which no malignant cell was found. Conclusion: This is the first report that the supernatants from FNA biopsy may contain tumor-derived exosomal RNA. These tumor-derived exosomes from FNA may provide a new liquid biopsy for the molecular diagnosis of cancer.

Characterization of the elemental and particle load of patient exhaled breath condensate and comparison with pulmonary lavages.

In the field of biomonitoring, exhaled breath condensate (EBC) is described as a potentially useful matrix for assessing inhalation exposure biomarkers in a non-invasive way. However, it is still unclear to what extent EBC is representative of the deep lung. To address this knowledge gap, EBC, bronchial washes (BWs), and bronchoalveolar lavages (BALs) were collected from 82 patients suffering from interstitial lung diseases (ILDs). The particulate contents and elemental composition of EBC, BW, and BAL were then compared in the same patients. The size distribution of particles in EBC was assessed with dynamic light scattering while inductively coupled plasma mass spectrometry was used to quantify its elemental composition. In addition, transmission electron microscopy coupled with energy dispersive x-ray spectrometry were used to further characterize samples of interest. EBC was found to be representative of both the sub-micron and nano-sized particle fractions of BAL and BW, with lower overall levels of elements in EBC than in BW and BAL. Silicon (Si) was the main component for all respiratory matrices with median levels of 2525µg l-1, 5643µg l-1and 5169µg l-1in the nano/ion fractions of EBC, BAL and BW, respectively. Moreover, Si levels in EBC from patients in this study were elevated compared to the levels reported in the literature for healthy subjects. Interestingly, Si levels in the EBC of ILD patients were inversely related to those in BAL and BW. In conclusion, the particulate content of EBC is associated with the lung particle burden and potentially correlates with pathologies, rendering it a relevant biomonitoring technique for the occupational and clinical fields.

Plastinated nasal model: a new concept of anatomically realistic cast.

BACKGROUND: For many years, researchers have been interested in investigating airflow and aerosol deposition in the nasal cavities. The nasal airways appear to be a complex geometrical system. Thus, in vitro experimental studies are frequently conducted with a more or less biomimetic nasal replica. AIM: This study is devoted to the development of an anatomically realistic nose model with bilateral nasal cavities, i.e. nasal anatomy, airway geometry and aerodynamic properties as close as possible to in vivo behaviour. METHODS: A specific plastination technique of cephalic extremities was developed by the Anatomy Laboratory at the Saint-Etienne University in the last 10 years. The plastinated models obtained were anatomically, geometrically and aerodynamically validated using several techniques (endoscopy, CT scans, acoustic rhinometry and rhinomanometry). RESULTS: Our plastination model exhibited a high level of anatomic quality, including a very good mucosa preservation. Aerodynamical and geometrical investigations highlighted a global behaviour of plastinated models perfectly in accordance with a nasal decongested healthy subject. CONCLUSIONS: The present plastination model provides a realistic cast of nasal airways, and may be a useful tool for nasal flow, drug delivery and aerosol deposition studies.

Improvement of malignant serous effusions diagnosis by quantitative analysis of molecular claudin 4 expression.

Claudin gene expression is frequently altered in human cancers. Our aim was to improve the cytology diagnosis of malignancy in serous fluids with the quantification of claudins compared with various classic molecular markers using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) method. Peritoneal or pleural effusions of 56 patients were assessed as malignant from histological analysis and 19 as benign. Claudin 4 was the most significantly upregulated (68%) marker in patients with malignant effusions. In cytologically negative malignant effusions, claudin 4 was found increased in 8/18 fluids. Quantitative RT-PCR is a sensitive method for the detection of free cancer cells in serous effusions.