An investigation of adequate volume for the diagnosis of malignancy in pleural fluids

S. C. Thomas, L. R. R. Davidson and M. E. McKean An investigation of adequate volume for the diagnosis of malignancy in pleural fluids Objective: The cytological examination of pleural effusions is an important investigation in the diagnosis of malignancy. Maximizing the chances of identifying malignant effusions is therefore desirable. Recent Royal College of Pathologists guidelines, based on the British Society for Clinical Cytology codes of practice, have suggested that a minimum of 20 ml of pleural fluid is required for diagnostic purposes. Methods & Results: We examined 2155 pleural fluids received over a 6‐year period in order to define a minimum required volume for adequacy. By examining the plateau phase of a graph of threshold volumes for initial samples received for each patient (n = 1584) we determine that a minimum fluid volume of 25 ml is required and that more than 50 ml does not improve sensitivity. Conclusion: Between 25 and 50 ml of fluid are required for the adequate assessment of pleural effusions for malignancy.     

Evaluation of serum and pleural levels of soluble B7-H4 in lung cancer patients with pleural effusion

Abstract

Objective: To evaluate the diagnostic value of sB7-H4 and CEA in both serum and pleural effusion of lung cancer patients.

Methods: Levels of sB7-H4 and CEA in 90 patients with malignant pleural effusion due to lung cancer and 58 patients with benign pleural effusion were measured by ELISA.

Results: The sB7-H4 and CEA levels in pleural effusion, serum and their ratio (F/S) were higher in lung cancer group than that in benign group (p?<?0.01). The diagnostic efficiency of sB7-H4 combined CEA was superior to either sB7-H4 or CEA.

Conclusions: Measurement of sB7-H4 and CEA might be useful diagnostic value for malignant effusion.     

Pyriproxyfen enhances the immunoglobulin G immune response in mice

Pyriproxyfen is a juvenile hormone mimic of vital importance for insect development with little risk to humans. This study was performed to investigate whether large doses of pyriproxyfen affect the immune response in mammals. Mice were immunized thrice with ovalbumin in 5% ethanol, with or without pyriproxyfen or alum. Large doses of pyriproxyfen (9 or 15 mM) significantly enhanced specific total IgG immune response. This enhancement was no longer present 24 hr after treatment with pyriproxyfen. These results suggest that pyriproxyfen is a safe chemical. Moreover, pyriproxyfen induced higher titers of IgG2a and enhanced tumor necrosis factor‐alpha and gamma‐interferon responses whereas alum induced IgG1 with enhanced interleukin‐4 and ‐10. These observations indicate that the mechanism of immune enhancement by pyriproxyfen may differ from that of alum.     

Sequence specificity of inter- and intramolecular G-quadruplex formation by human telomeric DNA

Human telomeric DNA consists of tandem repeats of the sequence 5'-d(TTAGGG)-3'. Guanine-rich DNA, such as that seen at telomeres, forms G-quadruplex secondary structures. Alternative forms of G-quadruplex structures can have differential effects on activities involved in telomere maintenance. With this in mind, we analyzed the effect of sequence and length of human telomeric DNA on G-quadruplex structures by native polyacrylamide gel electrophoresis and circular dichroism. Telomeric oligonucleotides shorter than four, 5'-d(TTAGGG)-3' repeats formed intermolecular G-quadruplexes. However, longer telomeric repeats formed intramolecular structures. Altering the 5'-d(TTAGGG)-3' to 5'-d(TTAGAG)-3' in any one of the repeats of 5'-d(TTAGGG)(4)-3' converted an intramolecular structure to intermolecular G-quadruplexes with varying degrees of parallel or anti-parallel-stranded character, depending on the length of incubation time and DNA sequence. These structures were most abundant in K(+)-containing buffers. Higher-order structures that exhibited ladders on polyacrylamide gels were observed only for oligonucleotides with the first telomeric repeat altered. Altering the sequence of 5'-d(TTAGGG)(8)-3' did not result in the substantial formation of intermolecular structures even when the oligonucleotide lacked four consecutive telomeric repeats. However, many of these intramolecular structures shared common features with intermolecular structures formed by the shorter oligonucleotides. The wide variability in structure formed by human telomeric sequence suggests that telomeric DNA structure can be easily modulated by proteins, oxidative damage, or point mutations resulting in conversion from one form of G-quadruplex to another.     

A comparative study of the effect of modification of the surface of human platelets on the receptors for aggregated immunoglobulins and for ristocetin-von Willebrand factor

The receptors for aggregated immunoglobulin G (IgG) (an Fc receptor) and for ristocetin-von Willebrand factor on human platelets were studied by means of various modifications of the platelet surface. The expression of these receptors was measured by the agglutination of platelets to ristocetin in the presence of von Willebrand factor, which is part of the factor VIII complex, and by the binding of aggregated IgG coupled to ³H-labelled diazobenzen. Treatment of platelets with chymotrypsin, trypsin, papain and pronase which removed protein and glycoprotein from the platelet under conditions where the release reaction was inhibited caused loss of the expression of the receptor for ristocetin-von Willebrand factor and an enhancement of that for aggregated IgG. Induction of membrane changes with ADP and of the release reaction with the ionophore A23187 abolished agglutination to ristocetin-von Willebrand factor but did not alter the receptor for aggregated IgG. Possible contributions of unspecific membrane changes, produced by protease treatment of platelets, to the modification of receptor expression were eliminated by the use of formaldehyde-treated platelets. Trypsin, papain and pronase destroyed the ability of these platelets to agglutinate to ristocetin-von Willebrand factor but produced no change in the binding of aggregated IgG. Therefore, the receptor for ristocetin-von Willebrand factor is truly sensitive to proteolysis while the Fc receptor is not, but is partially masked by protease-sensitive material.     

Proteomic differential display analysis identified upregulated astrocytic phosphoprotein PEA‐15 in human malignant pleural mesothelioma cell lines

We performed proteomic differential display analysis of human malignant pleural mesothelioma (MPM) cell lines and a human pleural mesothelial cell line by using 2‐DE and LC‐MS/MS. The human MPM cell lines were NCI‐H28, NCI‐H2052 and NCI‐H2452, and the human pleural mesothelial cell line was MeT‐5A. Between MeT‐5A and NCI‐H2052, we found 38 protein spots whose expression levels were different, from the results of 2‐DE; 28 protein spots appeared higher, and 10 other protein spots lower in NCI‐H2052 than in MeT‐5A. These spots were analyzed by LC‐MS/MS analysis and identified by a peptide sequence tag. However, from the results of 2‐DE of the other cell lines, there was only one consistently upregulated protein, astrocytic phosphoprotein PEA‐15, in all three MPM cell lines. Western blotting using specific antibodies against PEA‐15 confirmed the elevated expression level of PEA‐15 in all three MPM cell lines compared with MeT‐5A cells and normal pleura tissues from patients. PEA‐15 was knocked down in NCI‐H2052 cells, and the proliferation of PEA‐15‐silenced NCI‐H2052 cells was suppressed 7–15% compared with negative control cells. These results suggest that PEA‐15 expression is likely to be associated with the tumorigenesis of MPM.     

Fluorescence microscopy of mycobacteria in pleural effusions

The diagnostic advantage of fluorescence microscopy (FM) of Papanicolaou-stained cytological specimens obtained by bronchoscopy has been described previously. This study was designed to evaluate the method's diagnostic benefit in cytological preparations of pleural effusions in cases of active pulmonary tuberculosis. In contrast to bronchial material there is no advantage in cytological evaluation of pleural effusions by FM.     

“Patient-Like” nude mouse metastatic model of advanced human pleural cancer

Pleural Cancer in humans is a frequently occuring tumor, Recently, clinical ltrials have suggested that chemotherapy and immunotherapy administered intrapleually may elicit responses in early-stage diseases. However, atradiological and pleural endoscopic evaluation, most of the patients are found to have a visceral pleural involvement that is generally refractory to therapy and leads to a poor prognosis. The goal of this study was to construct a nude mouse model of human parietal- and visceral-pleural cancer that could reflect the clinical picture for this disease. The model could then be useful for drug discovery for pleural cancer. A well-differentiated human lung adenocarcinoma was used as intact tissue for implantation. Ten mice underwent parietal-pleural implantation and ten mice visceral-pleural implantation via a novel thoracotomy procedure we have developed. Symptoms of tumor growth were determined from weight loss, repiratory distress, or debilitation. Actual tumor growth and spread were measured at autopsy. The mouse survival curves of each group were estimated by the Kaplan-Meier Method and the difference of the median survial timje was assessed by the Log-rank test. The slopes of mean-mouse weight curves were compared using a standard two-sample t-test. 100% take rate was achieved in constructing the pleural cancer models. Tumor growth was initially assessed by symptomatology and survival: the median survival time was, repectively, 27.9 days 31 days for visceral-pleural and parietal-pleural implanted groups (P<0.05). The comparison between the slopes of the mean weight curves of corresponding groups demonstrated that visceral-pleural implanted animals lost significantly more weight than the parietal-pleural implanted animals (P <.001). Both in the visceral- and pariental-pleural implanted groups, post-mortem analysis revealed that tumor grew in all mic demonstrating local and regional spread mimicking clinical features. However, mediastinal lymph node metastases were observed only in mice with visceral pleural implantation. Patient-like models of human parietal-pleural and visceral-pleural cancer were constructed in nude mice using histologically ilntact human specimens. Tumor symptoms, growth, and spread as well as survival indicated that the parietal-pleural and visceral-pleural models represent, respectively, early-and advanced-stage disease. The “Patient-like” nude mouse models of pleural cancer now allow a rational basis for futher studies of pleural cancer biology, pathophysiology, and therapeutics.     

Long-term tissue cultures of human pleural effusions: A cytological follow-up

Summary A cytological follow-up was performed on long-term cultures of 53 metastatic pleural effusions and 34 cases of mesothelial hyperplasia. Cells were grown in Leighton tubes with up to 11 changes of cover slip in the same tube. Cell morphology and arrangement was followed for up to 1 year. Morphological cariteria are of no use in recognizing malignant cells with any degree of certainty. As to cell distribution, several types of heaping up are observed both in benign and malignant pleural effusions. In malignant cases, patches of cells in an epithelial-like arrangement are seen heaping up from their substrate. They can be recovered from the used medium by cytocentrifugation. Such behavior may explain the numerous unsuccessful attempts to establish epithelial cell lines, as the clusters of epithelial cells are progressively lost in the supernates. Supported in part by Contract INSERM ATP 76-1-493 and by a grant from the Ligue Nationale Fran?aise contre le Cancer.     

State-of-the-art cytology of pleural fluid,focusing on the diagnosis of mesothelioma

Mesothelioma has always been a challenging diagnosis to render in body cavity cytology samples. This review is a timely update on pleural fluid cytology and ancillary studies that should be considered in the diagnosis of mesothelial proliferations, specifically mesotheliomas. Information about new diagnostic approaches and ancillary studies in mesothelioma was obtained from the peer-reviewed literature and the authors' experiences. Although the morphological diagnosis of mesothelioma is fraught with numerous challenges given the overlap with other diagnostic entities, there are a variety of immunohistochemical and fluorescence in situ hybridization studies available to help in determining mesothelial origin and in distinguishing malignant proliferations from the more common benign or reactive mesothelial proliferations. Although ancillary studies can be helpful, there are important pitfalls to be aware of when interpreting these cases, and this review highlights some of the challenges that require caution.     

肺癌患者胸水游离氨基酸分析

本文对11例肺癌患者胸水13种游离氨基酸作了分析,并与28例正常人血浆游离氨基酸水平作了对照,结果表明:肺癌患者胸水的必需及非必需氨基酸普遍高于正常人血浆游离氨基酸,但其胸水谷氨酰胺水平则明显低于正常人血浆水平。     

Cytopathological spectrum of unusual malignant pleural effusions at a tertiary care centre in north India

BACKGROUND: Cytological examination of pleural fluid is one of the most informative laboratory procedures in the diagnosis of pleural effusions. Although tuberculosis is the commonest cause of pleural effusions in developing countries, tumours, including grade ones, can present with effusions. OBJECTIVE: The aim of the present study was to evaluate the uncommon causes of malignant pleural effusion. METHODS: A 2-year retrospective analysis of pleural fluid cytological specimens submitted to the Department of Cytopathology, PGIMER, Chandigarh between January 2003 and December 2004 was performed to retrieve unusual metastases. Out of a total of 898 samples reviewed, 710 were negative for malignancy and 24 cases were suspicious for malignancy. The remaining 164 cases were positive for malignancy, out of which 38 cases revealed malignancies other than adenocarcinoma. RESULTS: The 38 unusual malignancies metastasizing to the pleural cavity included 29 haematological malignancies (non-Hodgkin's lymphoma, acute lymphoid leukaemia, multiple myeloma and chronic myeloid leukaemia) and nine non-haematological malignancies (Ewing's sarcoma, neuroblastoma, Wilms' tumour, squamous cell carcinoma, small-cell carcinoma and malignant fibrous histiocytoma). CONCLUSION: Although metastatic adenocarcinoma was the commonest aetiology of malignant pleural effusions, a significant number of unusual causes of malignant pleural effusion were also encountered.     

Neurotensin expression and outcome of malignant pleural mesothelioma

Malignant pleural mesothelioma is a frequently fatal disease and the impact of available treatments is globally poor. Identification of new prognostic factors would help in the understanding of disease progression and, possibly, patient management. Here, we evaluate the prognostic impact of the neurotensin (NTS) and its cognate receptor (NTSR1) known for mediating cellular proliferation, survival, invasiveness, and mobility. We studied a series of 52 consecutive patients with epithelioid malignant mesothelioma undergoing management with curative intent, by immunohistochemistry for the expression of NTS and NTSR1. Specimens were scored as 0, 1, or 2 for less than 10%, between 10 and 50%, or more than 50% of NTS positive staining in tumor cells, respectively. Immunohistochemistry revealed that NTS and NTSR1 expression was found in 71.1% and 90.4% of malignant mesotheliomas, respectively. Using univariate analysis, expression of NTS was significantly (p = 0.015) related with a poor prognosis, with median survivals of 11.0 months, 18.4 months, and 29.8 months in patients showing expression scored as 2, 1, and 0, respectively. Multivariate analysis showed that expression of NTS (p = 0.007) and non-surgical therapy (p = 0.004) were independent predictors of poor prognosis. In order to evaluate the role of NTS/NTSR1 complex in mesothelioma progression, in vitro cell invasion assays and wound healing were performed on the mesothelioma cell line, MSTO-211H, and showed that inhibition of the NTS system resulted in a significant reduction of both migration and collagen invasion of mesothelioma cells. The expression of NTS is identified as a prognostic marker in patients with malignant pleural mesothelioma (Patent EP 08305971.7).     

Cul4A overexpression associated with Gli1 expression in malignant pleural mesothelioma

Malignant pleural mesothelioma (mesothelioma) is a highly aggressive cancer without an effective treatment. Cul4A, a scaffold protein that recruits substrates for degradation, is amplified in several human cancers, including mesothelioma. We have recently shown that Cul4A plays an oncogenic role in vitro and in a mouse model. In this study, we analysed clinical mesothelioma tumours and found moderate to strong expression of Cul4A in 70.9% (51/72) of these tumours, as shown by immunohistochemistry. In 72.2% mesothelioma tumours with increased Cul4A copy number identified by fluorescence in situ hybridization analysis, Cul4A protein expression was moderate to strong. Similarly, Cul4A was overexpressed and Cul4A copy number was increased in human mesothelioma cell lines. Because Gli1 is highly expressed in human mesothelioma cells, we compared Cul4A and Gli1 expression in mesothelioma tumours and found their expression associated (P < 0.05, chi‐square). In mesothelioma cell lines, inhibiting Cul4A by siRNA decreased Gli1 expression, suggesting that Gli1 expression is, at least in part, regulated by Cul4A in mesothelioma cells. Our results suggest a linkage between Cul4A and Gli1 expression in human mesothelioma.     

Selective determination of human immunoglobulin G by flow‐injection chemiluminescence

A novel flow‐injection chemiluminescence method was developed for the selective determination of human immunoglobulin G (IgG) in the presence of thiomersal by changing the flow rates of peristaltic pump. The study was based on the independence and additivity of the CL signals of human IgG and thiomersal in the galangin–potassium permanganate–polyphosphoric acid system. In meantime, two equations relating to the concentrations of mixing solutions of human IgG and thiomersal vs the CL intensity were established and solved, on the basis of which the content of thiomersal included in samples was simultaneously determined too. The enhanced CL intensity was in proportion to concerntrations in the range 8.0 × 10^?7 to 8.0 × 10^?5 g/mL for human IgG and 1.0 × 10^?7 to 2.0 × 10^?6 g/mL for thiomersal with the detection limits of 5.0 × 10^?7 g/mL for human IgG and 6.0 × 10^?8 g/mL for thiomersal, respectively. The relative standard deviation for 1.0 × 10^?5 g/mL human IgG was 0.8% and for 2.0 × 10^?7 g/mL thiomersal it was 2.0% (n = 10). The proposed method was applied to determine three synthetic samples with recoveries of 91.5–109.5%. In addition, the possible chemiluminescence mechanisms are discussed as well. Copyright © 2010 John Wiley & Sons, Ltd.     

The relationship between airway immunoglobulin activity and eosinophils in COPD

In chronic obstructive pulmonary disease (COPD), the effects of inhaled corticosteroids are predicted by blood eosinophil counts. We previously briefly reported increased immunoglobulin (Ig)A and IgM levels in bronchoalveolar lavage (BAL) of COPD patients with higher (eosinophil^high) compared to lower (eosinophil^low) blood eosinophils (>250/μL versus < 150/μL), suggesting differences in adaptive immune function. An inverse relationship exists between eosinophil counts and airway pathogenic bacteria levels. The mechanistic reasons for these associations between eosinophils, corticosteroids and pathogenic bacteria are unclear. IgA, IgM and IgG levels were assessed in BAL, bronchial biopsies and epithelium collected from eosinophil^high (n = 20) and eosinophil^low (n = 21) patients. Bronchial B-cell numbers were measured by immunohistochemistry. B-cell activity was assessed in bronchial samples and following exposure to BAL from eosinophil^high and eosinophil^low patients. BAL levels of non-typeable Haemophilus influenza (NTHi)-specific immunoglobulins were quantified. Results showed airway expression of IgA, IgG1 and IgM were lower in eosinophil^low compared to eosinophil^high patients, with lower levels of NTHi-specific IgA and IgM. Bronchial B-cell numbers were similar in both groups, but B-cell activity was lower in eosinophil^low patients. In conclusion, COPD eosinophil^low patients show differences in adaptive immune function compared to COPD eosinophil^high patients. These differences may cause different microbiomes in these COPD phenotypes.     

Inhibition of yes‐associated protein down‐regulates PD‐L1 (CD274) expression in human malignant pleural mesothelioma

Although tumour PD‐L1 (CD274) expression had been used as a predictive biomarker in checkpoint immunotherapy targeting the PD1/PD‐L1 axis in various cancers, the regulation of PD‐L1 (CD274) expression is unclear. Yes‐associated protein (YAP), an important oncogenic protein in Hippo signalling pathway, reportedly promotes cancer development. We investigated whether inhibition of YAP down‐regulates PD‐L1 (CD274) in human malignant pleural mesothelioma (MPM). Western blotting showed that 2 human MPM cell lines (H2052 and 211H) had increased PD‐L1 protein expression compared to H290, MS‐1 and H28 cells. In H2052 and 211H cells, PD‐L1 mRNA expression was significantly increased compared to other MPM cell lines; YAP knockdown by small interfering RNA decreased PD‐L1 protein and mRNA expression. Forced overexpression of the YAP gene increased PD‐L1 protein expression in H2452 cells. Chromatin immunoprecipitation (ChIP) assay showed the precipitation of PD‐L1 enhancer region encompassing 2 putative YAP‐TEAD‐binding sites in H2052 cells. We found that, in human MPM tissue microarray samples, YAP and PD‐L1 concurrently expressed in immunohistochemistry stain (n = 70, P < .05, chi‐square). We conclude that PD‐L1 is correlated with YAP expression, and inhibition of YAP down‐regulates PD‐L1 expression in human MPM. Further study of how YAP regulates PD‐L1 in MPM is warranted.