Quiz case: Abnormal haematopoietic cells in pleural effusion.

This case was presented because of the number of plasmablasts in a patient with a medical history of multiple myeloma. Flow cytometry is a "gold standard" technique for the diagnosis of haematological malignancies. This technique works for all fluids and should be performed in effusions (pleural, pericardial, ascites) in cases of suspected haematological malignancy. Alternatively, immunohistochemistry using appropriate markers could be performed if flow cytometry is not available. This case illustrates a pleural infiltration by plasmablasts. Myelomatous cells were characterised by immunocytochemistry and flow cytometry.

Cytological Samples: An Asset for the Diagnosis and Therapeutic Management of Patients with Lung Cancer.

BACKGROUND: Lung cancer has become the leading cause of cancer death for men and women. Most patients are diagnosed at an advanced stage when surgery is no longer a therapeutic option. At this stage, cytological samples are often the less invasive source for diagnosis and the determination of predictive markers. We assessed the ability of cytological samples to perform diagnosis, and to establish molecular profile and PD-L1 expression, which are essential for the therapeutic management of patients. METHODS: We included 259 cytological samples with suspected tumor cells and assessed the ability to confirm the type of malignancy by immunocytochemistry. We summarized results of molecular testing by next generation sequencing (NGS) and PD-L1 expression from these samples. Finally, we analyzed the impact of these results in the patient management. RESULTS: Among the 259 cytological samples, 189 concerned lung cancers. Of these, immunocytochemistry confirmed the diagnosis in 95%. Molecular testing by NGS was obtained in 93% of lung adenocarcinomas and non-small cell lung cancer. PD-L1 results were obtained in 75% of patients tested. The results obtained with cytological samples led to a therapeutic decision in 87% of patients. CONCLUSION: Cytological samples are obtained by minimally invasive procedures and can provide enough material for the diagnosis and therapeutic management in lung cancer patients.

miR-140-5p and miR-140-3p: Key Actors in Aging-Related Diseases?

microRNAs (miRNAs) are small single strand non-coding RNAs and powerful gene expression regulators. They mainly bind to the 3'UTR sequence of targeted mRNA, leading to their degradation or translation inhibition. miR-140 gene encodes the pre-miR-140 that generates the two mature miRNAs miR-140-5p and miR-140-3p. miR-140-5p/-3p have been associated with the development and progression of cancers, but also non-neoplastic diseases. In aging-related diseases, miR-140-5p and miR-140-3p expressions are modulated. The seric levels of these two miRNAs are used as circulating biomarkers and may represent predictive tools. They are also considered key actors in the pathophysiology of aging-related diseases. miR-140-5p/-3p repress targets regulating cell proliferation, apoptosis, senescence, and inflammation. This work focuses on the roles of miR-140-3p and miR-140-5p in aging-related diseases, details their regulation (i.e., by long non-coding RNA), and reviews the molecular targets of theses miRNAs involved in aging pathophysiology.

Comparison between Immunocytochemistry, FISH and NGS for ALK and ROS1 Rearrangement Detection in Cytological Samples.

The detection of ROS1 and ALK rearrangements is performed for advanced-stage non-small cell lung cancer. Several techniques can be used on cytological samples, such as immunocytochemistry (ICC), fluorescence in situ hybridization (FISH) and, more recently, next-generation sequencing (NGS), which is gradually becoming the gold standard. We performed a retrospective study to compare ALK and ROS1 rearrangement results from immunocytochemistry, FISH and NGS methods from 131 cytological samples. Compared to NGS, the sensitivity and specificity of ICC were 0.79 and 0.91, respectively, for ALK, and 1 and 0.87 for ROS1. Regarding FISH, the sensitivity and specificity were both at 1 for ALK and ROS1 probes. False-positive cases obtained by ICC were systematically corrected by FISH. When using ICC and FISH techniques, results are very close to NGS. The false-positive cases obtained by ICC are corrected by FISH, and the true-positive cases are confirmed. NGS has the potential to improve the detection of ALK and ROS1 rearrangements in cytological samples; however, the cost of this technique is still much higher than the sequential use of ICC and FISH.

CD146 at the Interface between Oxidative Stress and the Wnt Signaling Pathway in Systemic Sclerosis.

CD146 involvement was recently described in skin fibrosis of systemic sclerosis through its regulation of the Wnt pathway. Because the interaction between Wnt and ROS signaling plays a major role in fibrosis, we hypothesized that in systemic sclerosis, CD146 may regulate Wnt/ROS crosstalk. Using a transcriptomic and western blot analysis performed on CD146 wild-type or knockout mouse embryonic fibroblasts, we showed a procanonical Wnt hallmark in the absence of CD146 that is reversed when CD146 expression is restored. We found an elevated ROS content in knockout cells and an increase in DNA oxidative damage in the skin sections of knockout mice compared with those of wild-type mice. We also showed that ROS increased CD146 and its noncanonical Wnt ligand, WNT5A, only in wild-type cells. In humans, fibroblasts from patients with systemic sclerosis presented higher ROS content and expressed CD146, whereas control fibroblasts did not. Moreover, CD146 and its ligand were upregulated by ROS in both human fibroblasts. The increase in bleomycin-induced WNT5A expression was abrogated when CD146 was silenced. We showed an interplay between Wnt and ROS signaling in systemic sclerosis, regulated by CD146, which promotes the noncanonical Wnt pathway and prevents ROS signaling, opening the way for innovative therapeutic strategies.

miR-376a-3p and miR-376b-3p overexpression in Hutchinson-Gilford progeria fibroblasts inhibits cell proliferation and induces premature senescence.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder, in which an abnormal and toxic protein called progerin, accumulates in cell nuclei, leading to major cellular defects. Among them, chromatin remodeling drives gene expression changes, including miRNA dysregulation. In our study, we evaluated miRNA expression profiles in HGPS and control fibroblasts. We identified an enrichment of overexpressed miRNAs belonging to the 14q32.2-14q32.3 miRNA cluster. Using 3D FISH, we demonstrated that overexpression of these miRNAs is associated with chromatin remodeling at this specific locus in HGPS fibroblasts. We then focused on miR-376b-3p and miR-376a-3p, both overexpressed in HGPS fibroblasts. We demonstrated that their induced overexpression in control fibroblasts decreases cell proliferation and increases senescence, whereas their inhibition in HGPS fibroblasts rescues proliferation defects and senescence and decreases progerin accumulation. By targeting these major processes linked to premature aging, these two miRNAs may play a pivotal role in the pathophysiology of HGPS.

The Role of the Adhesion Receptor CD146 and Its Soluble Form in Human Embryo Implantation and Pregnancy.

CD146 is an adhesion molecule essentially located in the vascular system, which has been described to play an important role in angiogenesis. A soluble form of CD146, called sCD146, is detected in the bloodstream and is known as an angiogenic factor. During placental development, CD146 is selectively expressed in extravillous trophoblasts. A growing body of evidence shows that CD146 and, in particular, sCD146, regulate extravillous trophoblasts migration and invasion both in vitro and in vivo. Hereby, we review expression and functions of CD146/sCD146 in the obstetrical field, mainly in pregnancy and in embryo implantation. We emphasized the relevance of quantifying sCD146 in the plasma of pregnant women or in embryo supernatant in the case of in vitro fertilization (IVF) to predict pathological pregnancy such as preeclampsia or implantation defect. This review will also shed light on some major results that led us to define CD146/sCD146 as a biomarker of placental development and paves the way toward identification of new therapeutic targets during implantation and pregnancy.

Pharmacokinetics and pharmacogenetics of liposomal cytarabine in AML patients treated with CPX-351.

CPX-351 is a liposome encapsulating cytarabine and daunorubicin for treating Acute Myeloid Leukemia (AML) patients. To what extent differences in cytidine deaminase (CDA) activity, the enzyme that catabolizes free cytarabine in the liver, can affect the pharmacokinetics of liposomal cytarabine as well, is unknown. We have studied the pharmacokinetics (PK) of released, liposomal and total cytarabine using a population-modeling approach in 9 adult AML patients treated with liposomal CPX-351. Exposure levels and PK parameters were compared with respect to the patient's CDA status (i.e., Poor Metabolizer (PM) vs. Extensive Metabolizer (EM)). Overall response rate was 75%, and 56% of patients had non-hematological severe toxicities, including one lethal toxicity. All patients had febrile neutropenia. A large (>60%) inter-individual variability was observed on pharmacokinetics parameters and subsequent drug levels. A trend towards severe toxicities was observed in patients with higher exposure of cytarabine. Results showed that liposomal CPX-351 led to sustained exposure with reduced clearance (Cl = 0.16 L/h) and prolonged half-life (T1/2 = 28 h). Liposomal nanoparticles were observed transiently in bone marrow with cytarabine levels 2.3-time higher than in plasma. Seven out of 9 patients were PM with a strong impact on the PK parameters, i.e., PM patients showing higher cytarabine levels as compared with EM patients (AUC: 5536 vs. 1784 ng/mL.h), sustained plasma exposure (T1/2: 33.9 vs. 13.7 h), and reduced clearance (Cl: 0.12 vs. 0.29 L/h). This proof-of-concept study suggests that CDA status has a major impact on cytarabine PK and possibly safety in AML patients even when administered as a liposome.

Immunocytochemical Detection of ALK and ROS1 Rearrangements in Lung Cancer Cytological Samples.

The detection of molecular alterations such as ROS1 and ALK rearrangements is performed as part of the diagnosis of advanced-stage lung adenocarcinoma. These alterations allow the treatments with tyrosine kinase inhibitors. Cytological samples are very useful as up to 40% patients are diagnosed with this type of sample. Here we describe the immunocytochemistry technique usable to reveal the overexpression of ALK or ROS1 tyrosine kinase receptors secondary to ALK and ROS1 rearrangements, respectively.

miR-9 Does Not Regulate Lamin A Expression in Metastatic Cells from Lung Adenocarcinoma.

In lung adenocarcinoma, low lamin A expression in pleural metastatic cells has been proposed as a pejorative factor. miR-9 physiologically inhibits the expression of lamin A in neural cells and seems to be a central actor in the carcinogenesis and the metastatic process in lung cancer. Thus, it could be a good candidate to explain the reduction of lamin A expression in lung adenocarcinoma cells. miR-9 expression was analyzed in 16 pleural effusions containing metastatic cells from lung adenocarcinoma and was significantly reduced in patients from the 'Low lamin A expression' group compared to patients from the 'High lamin A expression' group. Then, carcinoma cells selection by fluorescence-activated cell sorting (FACS) was performed according to epithelial membrane antigen (EMA) expression, reflecting lamin A expression. miR-9 was underexpressed in lamin A- carcinoma cells compared to lamin A+ carcinoma cells in patients from the 'Low lamin A expression' group, whereas there was no difference of miR-9 expression between lamin A+ and lamin A- carcinoma cells in patients from the 'High lamin A expression' group. These results suggest that miR-9 does not regulate lamin A expression in metastatic cells from lung adenocarcinoma. On the contrary, miR-9 expression was shown to be reduced in lamin A-negative carcinoma cells.

[Detection of ALK and ROS1 rearrangements by immunocytochemistry on cytological samples]. / Mise en évidence du réarrangement d'ALK et ROS1 en immunocytochimie sur liquides de ponction.

The identification of ALK and ROS1 rearrangements has become essential for the theranostic management of patients with non-small cell lung cancer, especially in stage IV or inoperable patients. These testings are now performed by immunohistochemistry on histological samples and confirmed by fluorescent in situ hybridization in case of positive or doubtful results. The diagnosis of lung cancer is often performed at an advanced or metastatic stage and cytological sample could be the only material containing malignant cells available at these stages. Therefore, the detection of ALK and ROS1 rearrangement by immunocytochemical analysis on cytological specimens is needed. We performed this test on 27 cytological samples of lung adenocarcinomas, and we compared our results with several other techniques: on the same sample or on biopsy in another laboratory, on the same sample by fluorescent in situ hybridization and/or immunochemistry. We found a very good concordance between all these techniques, thus validating our immunocytochemical method on cytological samples according to the ISO 15189 norm.

Lamins in Lung Cancer: Biomarkers and Key Factors for Disease Progression through miR-9 Regulation?

Lung cancer represents the primary cause of cancer death in the world. Malignant cells identification and characterization are crucial for the diagnosis and management of patients with primary or metastatic cancers. In this context, the identification of new biomarkers is essential to improve the differential diagnosis between cancer subtypes, to select the most appropriate therapy, and to establish prognostic correlations. Nuclear abnormalities are hallmarks of carcinoma cells and are used as cytological diagnostic criteria of malignancy. Lamins (divided into A- and B-types) are localized in the nuclear matrix comprising nuclear lamina, where they act as scaffolding protein, involved in many nuclear functions, with regulatory effects on the cell cycle and differentiation, senescence and apoptosis. Previous studies have suggested that lamins are involved in tumor development and progression with opposite results concerning their prognostic role. This review provides an overview of lamins expression in lung cancer and the relevance of these findings for disease diagnosis and prognosis. Furthermore, we discuss the link between A-type lamins expression in lung carcinoma cells and nuclear deformability, epithelial to mesenchymal transition, and metastatic potential, and which mechanisms could regulate A-type lamins expression in lung cancer, such as the microRNA miR-9.

Chest ultrasonography to assess the kinetics and efficacy of talc pleurodesis in a model of pneumothorax: an experimental animal study.

Talc pleurodesis is used to avoid recurrences in malignant pleural effusions or pneumothorax. The lack of lung sliding detected by chest ultrasonography (CUS) after talc application is indicative of the effectiveness of pleurodesis. The objective of our study was to explore, in an animal model, the capacity of CUS to predict the quality of a symphysis induced by talc poudrage (TP) and talc slurry (TS). We induced an artificial pneumothorax in six healthy pigs prior to talc application. TP was performed on one hemithorax, followed by TS on the other side 1 week later. 108 points on the chest were marked and evaluated by ultrasonography during the study. TP showed higher sonographic scores compared to TS starting from 72 h after talc administration. At autopsy, a higher grade of symphysis was observed for TP, and a high correlation rate was registered between CUS and macroscopic findings. Histological analysis also showed a higher grade of pleural symphysis for TP. CUS is a reliable tool to assess talc pleurodesis. The quality and kinetics of the pleural symphysis are also evaluable by ultrasonography. Pleurodesis by TP is more effective than TS in this experimental model of pneumothorax.