Heparanase expression: a potential ancillary diagnostic tool for distinguishing between malignant cells and reactive mesothelium in body cavity effusions

OBJECTIVE: Heparanase, an endoglycosidase that cleaves heparan sulphate, is frequently expressed in carcinomas and was suggested to play a role in cell invasion and metastasis. We investigated whether heparanase expression may serve as a reliable marker to discriminate benign mesothelial cells from malignant cells shed into body cavities. METHODS AND RESULTS: Cytological smears of effusions from 51 hospitalized patients were immunostained for heparanase. Strong immunoreactivity was noted in 35 of 40 (88%) carcinoma samples and in all three malignant mesothelioma cases. Only rare (<3%) reactive mesothelial cells were noted showing a faint negligible staining. Specificity was 100%, sensitivity 88%, and positive and negative predictive values were 100% and 89% respectively. CONCLUSIONS: Our results suggest that heparanase may be of value as a complementary component in a diagnostic panel of markers, contributing to its reliability and accuracy.     

Imaging and therapy of malignant pleural mesothelioma using replication-competent herpes simplex viruses

BACKGROUND: Malignant pleural mesothelioma (MPM) is an aggressive cancer that is refractory to current treatment modalities. Oncolytic herpes simplex viruses (HSV) used for gene therapy are genetically engineered, replication-competent viruses that selectively target tumor cells while sparing normal host tissue. The localized nature, the potential accessibility and the relative lack of distant metastasis make MPM a particularly suitable disease for oncolytic viral therapy. METHODS: The infectivity, selective replication, vector spread and cytotoxic ability of three oncolytic HSV: G207, NV1020 and NV1066, were tested against eleven pathological types of MPM cell lines including those that are resistant to radiation therapy, gemcitabine or cisplatin. The therapeutic efficacy and the effect on survival of NV1066 were confirmed in a murine MPM model. RESULTS: All three oncolytic HSV were highly effective against all the MPM cell lines tested. Even at very low concentrations of MOI 0.01 (MOI: multiplicity of viral infection, ratio of viral particles per cancer cell), HSV were highly effective against MPM cells that are resistant to radiation, gemcitabine and cisplatin. NV1066, an oncolytic HSV that expresses green fluorescent protein (GFP), was able to delineate the extent of the disease in a murine model of MPM due to selective infection and expression of GFP in tumor cells. Furthermore, NV1066 was able to reduce the tumor burden and prolong survival even when treatment was at an advanced stage of the disease. CONCLUSION: These findings support the continued investigation of oncolytic HSV as potential therapy for patients with therapy-resistant MPM.     

Discriminant analysis of image karyometric variables in benign and malignant melanocytic skin lesions

OBJECTIVE: To perform a quantitative analysis to identify which of 7 nuclear morphometry-related variables are of diagnostic value in distinguishing benign from malignant melanocytic skin lesions. STUDY DESIGN: At the Institute of Pathology, University of Nis, formalin-fixed, paraffin-embedded skin biopsies from 23 cases of benign nevi (18 intradermal and 5 junctional) and 25 cases of primary nodular malignant melanomas were retrieved. Specimens were routinely stained with hematoxylin and eosin and analyzed using a computer-assisted interactive image analysis system. Nuclear area, equivalent diameter, volume of equivalent sphere, perimeter, mean chord, circularity and integrated optical density were estimated after manual editing of binary images. RESULTS: In univariate analysis, 6 features were found to be significantly different between the benign and malignant groups (P < .0001); all measured nuclear variables (except circularity) were higher in malignant melanomas. No significant differences were found among lesions with respect to nuclear shape. Using discriminant function analysis, a correct diagnosis was achieved in 95.8% of benign nevi cases and 84.0% of malignant melanoma cases. The best discriminant variable was nuclear area. CONCLUSION: Image analysis is diagnostically relevant to the evaluation of melanocytic lesions of the skin. The area of the nucleus appeared to have potential for differentiating benign from malignant tumors and can be estimated in the course of routine histology.     

Ultrastructure of pleural mesothelioma and pulmonary adenocarcinoma in malignant effusions as compared with reactive mesothelial cells.

OBJECTIVE: To determine the ultrastructural features of diffuse malignant pleural mesothelioma cells in cytologic specimens from pleural effusions. STUDY DESIGN: We retrospectively studied 35 pleural effusions: 12 diffuse malignant pleural mesotheliomas (8 epithelial type, 4 biphasic type), 12 pulmonary adenocarcinomas and 11 cases of reactive mesothelial cells. RESULTS: In the cytoplasm, reactive and malignant mesothelial cells had more-abundant intermediate filaments (P < .05, P < .01) and fewer free ribosomes (P < .001, P < .001) than adenocarcinoma cells. Reactive mesothelial cells had fewer mitochondria than mesothelioma cells (P < .05). Mesothelioma cells had longer, thinner microvilli on the cell surfaces (P < .001); length/diameter ratios of microvilli were 19.1 +/- 7.0 (mesothelioma) vs. 9.1 +/- 2.2 (adenocarcinoma) and 9.2 +/- 2.4 (mesothelial cells). Giant intercellular junctions (desmosomes or desmosomelike structures > 1 micron in length) were found in eight cases of mesothelioma. Core filaments or rootlets in microvilli were present in two cases of adenocarcinoma. CONCLUSION: Because cytologic specimens from pleural effusions were easy to obtain, we think ultrastructural cytology is useful in distinguishing mesothelioma from adenocarcinoma and benign effusions.     

Diagnostic value of GLUT-1 in distinguishing malignant mesothelioma from reactive mesothelial cells: a meta-analysis

Abstract

Background: Previous studies have demonstrated the diagnostic value of glucose transporter 1 (GLUT-1) to distinguish malignant mesothelioma (MM) from reactive mesothelial cells (RMC), but the results are inconsistent. The purpose of this meta-analysis is to investigate the diagnostic accuracy of GLUT-1 in distinguishing MM from RMC.

Methods: A systematical search was conducted until May 2019 in PubMed, Medline, Embase and the Cochrane Library. The revised tool for the quality assessment of diagnostic accuracy studies (QUADAS-2) was used to assess the quality of the eligible studies. The Stata15 and Review Manager5.3 software programmes were used to perform the meta-analysis.

Results: A total of 24 studies, including 969?MM patients and 1080 RMC individuals were explored in the meta-analysis. The summary assessments revealed that the pooled sensitivity was 0.73 (95% CI, 0.62–0.81) and the pooled specificity was 0.95 (95% CI, 0.91–0.98). The area under the summary ROC curve (AUC) was 0.93 (95% CI: 0.91–0.95).

Conclusions: GLUT-1 is highly accurate to distinguish MM from RMC.     

Computer-assisted differential diagnosis of malignant mesothelioma based on syntactic structure analysis.

BACKGROUND: Malignant mesothelioma, a mesoderm-derived tumor, is related to asbestos exposure and remains a diagnostic challenge because none of the genetic or immunohistochemical markers have yet been proven to be specific. To assist in the identification of mesothelioma and to differentiate it from other common lesions at the same location, we have tested the performance of syntactic structure analysis (SSA) in an automated classification procedure. MATERIALS AND METHODS: Light-microscopic images of tissue sections of malignant mesothelioma, hyperplastic mesothelium, and adenocarcinoma were analyzed using parameters selected from the Voronoi diagram, Gabriel's graph, and the minimum spanning tree which were classified with a K-nearest-neighbor algorithm. RESULTS: Results showed that mesotheliomas were diagnosed correctly in 74% of the cases; 76% of the adenocarcinomas were correctly graded, and 88% of the mesotheliomas were correctly typed. The performance of the parameters was dependent on the obtained classification (i.e., tumor-tumor versus tumor-benign). CONCLUSIONS: Our results suggest that SSA is valuable in the differential classification of mesothelioma and that it supplements a visually appraised diagnosis. The recognition scores may be increased by a combination of SSA with, for example, cellular or nuclear parameters, measured at higher magnifications to form a solid base for fully automated expert systems.     

High-performance capillary electrophoretic analysis of hyaluronan in effusions from human malignant mesothelioma

A procedure to quantify hyaluronan in effusions from human malignant mesothelioma using a highly sensitive and reproducible high-performance capillary electrophoresis (HPCE) method is presented. Following ethanol precipitation, hyaluronan and galactosaminoglycans were degraded to Δ^4.5-dissacharides with a mixture of chondroitinases ABC and AC. Heparan sulphate and proteins/glycoproteins were separated by ultrafiltration on a Centricon 3 membrane, and hyaluronan-derived disaccharides were analysed by direct injection of the filtrate into a HPCE system. Determination of hyaluronan in effusions from five healthy individuals and three patients with mesothelioma gave values comparable to those found using the HPLC method. One of the advantages of the HPCE method as compared to HPLC is the low solvent consumption. The much lower detection limit (attomole level) of the HPCE method may also allow the analysis of hyaluronan content in serum. The contribution of HPCE in diagnosis of a neoplasm, such as human malignant mesothelioma, illustrates the great potential of this technique in the field of life sciences.     

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).     

Discriminant analysis of event-related potential curves using smoothed principal components

Principal component analysis enhanced by the use of smoothing is used in conjunction with discriminant analysis techniques to devise a statistical classification method for the analysis of event-related potential data. A training set of premedication potentials collected from adolescents with attention-deficit hyperactive disorder (ADHD) is used to predict whether adolescents from an independent subject group will respond to long-term medication. Comparison of outcome prediction rates demonstrates that this method, which uses information from the whole ERP curve, is superior to the classification technique currently used by clinicians, which is based on a single ERP curve feature. The need to administer an initial dose of medication to classify patients is also eliminated.     

Fine needle aspiration cytology of malignant mesothelioma

The results of fine needle aspiration (FNA) cytology in 19 cases of malignant mesothelioma are presented. Adequate material for a diagnosis of malignancy was obtained in 17 cases, and in 8 cases a specific diagnosis of mesothelioma could be made. In four other cases, the findings were either consistent with or suggestive of mesothelioma; in four, accurate distinction from other neoplasms was not possible, and in two cases, adenocarcinoma was suggested. The spectrum of cytologic findings ranged from neoplasms of purely epithelial appearance through more pleomorphic biphasic neoplasms to anaplastic tumors. A combination of epithelial-like cell clusters, pavement-like sheets of epithelial cells with well-defined cell borders and prominent cell separation, dispersed angular cells with dense cytoplasm and some spindle-cell forms was the most specific cytologic pattern for mesothelioma. In four neoplasms, ultrastructural examination of aspirated material provided the additional evidence for a definitive diagnosis. The identification of hyaluronic acid within intracytoplasmic vacuoles, either in smears or in cell blocks, confirmed the diagnosis in three tumors. Only in one case, with a strong clinical background suggesting mesothelioma, was the cytologic preparation sufficient for diagnosis without ancillary diagnostic methods. FNA is of particular value in the diagnosis of pleural mesothelioma in patients who do not present with a pleural effusion. Obtaining material for cell block preparations, cytochemistry or ultrastructural study is generally necessary for definitive tumor typing.     

Establishment of a human malignant fibrous mesothelioma cell line and the biological characteristics compared with malignant epithelial mesothelioma cell line

Two human malignant mesothelioma cell lines, which we designated "epithelial mesothelioma cells" and "fibrous mesothelioma cells", were established from the pleural fluid containing malignant mesothelial cells of a 72-year-old Japanese man. These cell lines were separated by the colonial techniques from the initiation of the primary cultures and grew well without interruption for 12 years. They were characterized as producing hyaluronic acid. These cell lines displayed different biological characteristics, including morphology, heterotransplantability and genetics using with BAC array CGH. The epithelial mesothelioma cells were epithelial in shape and transplantable into the subcutis of nude mice, while the cells of the fibrous mesothelioma line were fibroblast-like and transplantable into the submucosa of Hamster's cheek pouches but not into the subcutis of nude mice. The mesotheliomas are classified into three types: epithelial mesothelioma, fibrous mesothelioma and mixed type. The gene copy number losses observed on 9p21.3, 9p21.2, 9p21.1, among others may be a major mechanism of malignant mesothelioma carcinogenesis. We considered and supported the combination theory for the histogenesis of malignant mesothelioma.     

Early diagnosis of mesothelioma in serous effusions using AgNOR analysis

OBJECTIVE: To compare the results of conventional cytology, DNA image cytometry, immunocytochemistry and argyrophilic nucleolar organizer region (AgNOR) analysis for the diagnosis of malignant cells in serous effusions. STUDY DESIGN: One hundred twenty effusions, 40 with carcinoses, 40 with malignant mesotheliomas and 40 without tumor cells on follow-up were studied by conventional cytology and three adjunctive methods. RESULTS: Unequivocal tumor cells were detected in 92.5% of effusions due to carcinoses and in 45% due to mesotheliomas. Applying immunocytochemistry with BerEP-4 positivity and DNA image cytometry with aneuploidy as a marker revealed 100% of carcinoses and 71.7% of mesotheliomas. Applying the experimentally found thresholds of 2.5 AgNORs as "satellites" and 4.5 AgNORs as "satellites and clusters" together as mean values per nucleus resulted in a 95% correct rate of mesothelioma and 100% rate of carcinoma cell identification without false positive diagnoses. CONCLUSION: AgNOR analysis may be a useful adjunct to other methods in the routine diagnosis of malignant serous effusions. It seems to be the most sensitive method in early cytologic diagnosis of mesotheliomas in effusions. Seventy-three percent of malignant mesotheliomas were diagnosed cytologically at first on effusions. Forty-seven percent of patients with malignant mesotheliomas were identified at the early tumor stage T1 N0 M0.     

Cytologic presentation of malignant mesothelioma in pleural effusions

1,601 pleural effusions were found to be malignant between 1976 and 1987. Among these were 26 (1.6% of the malignant effusions) mesothelioma. Only 2 cases showed pronounced cytologic features that made a definite diagnosis possible on cytologic criteria alone. In 20 cases diagnosis of mesothelioma was strongly suggested by the patient's history and cytology of the effusion was compatible with mesothelioma. In the other 4 cases special examinations (histo- and immunohistochemistry, electron microscopy) led to the final diagnosis. The cytologic features of mesothelioma and other examination techniques, needed to resolve the differential diagnosis of mesothelioma versus other neoplasm in pleural effusions, are discussed.     

Pleural malignant mesothelioma, genetic susceptibility and asbestos exposure

Pleural malignant mesothelioma (MM) is a rare but extremely aggressive cancer. The limited impact of standard therapeutic treatments on survival rates makes the identification of factors that increase the individual risk a leading priority. The high proportion of cases explained by exposure to asbestos has guided intervention policies to an effective ban of this compound from our environment. However, MM cannot be solely attributed to this agent, and the role of predisposing factors and their interaction with asbestos exposure is increasingly studied. The role of mEH, GSTM1, GSTT1, NAT2, and CYP1A1 genotypes in modulating susceptibility to MM was examined in a case-control study of 80 subjects with a confirmed diagnosis of MM and 255 controls. Subjects with low mEH activity showed a significantly increased risk of MM (OR, 2.51; 95% CI, 1.11-5.68). The association was stronger in the group with low asbestos exposure (OR, 7.83; 95% CI, 0.98-62.60). A significant increased risk of MM was also found in NAT2 fast acetylators (OR, 1.74; 95% CI, 1.02-2.96). The presence of synergisms between genotypes, i.e., mEH and NAT2 (LRT for heterogeneity p<0.023), mEH and GSTM1 (LRT p<0.061), and NAT2 and GSTM1 (LRT p<0.049), combined with the interaction observed with exposure to asbestos, suggests the presence of gene-environment and gene-gene interactions in the development of MM, although the size of the study group does not allow to draw clearcut conclusions. Since genetic polymorphisms can also modify the extent of genetic damage occurring in subjects exposed to carcinogens, we measured the frequency of micronuclei in peripheral blood lymphocytes of a subgroup of MM cases. The limited number of cases (28) did not allow to observe significant effects. In conclusion, these results strengthen the hypothesis that individual susceptibility to MM can be modulated by the interaction between polymorphic genes involved in the metabolism and the intensity of asbestos exposure.     

Immune responses and immunotherapeutic interventions in malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive, primary pleural malignancy with poor prognosis, hypothesized to originate from a chronic inflammatory state within the pleura. Similar to what has been observed in other solid tumors (melanoma, ovarian and colorectal cancer), clinical and pre-clinical MPM investigations have correlated anti-tumor immune responses with improved survival. As such, a better understanding of the complex MPM tumor microenvironment is imperative in strategizing successful immunotherapies. Herein, we review the immune responses vital to the development and progression of MPM, as well as assess the role of immunomodulatory therapies, highlighting recent pre-clinical and clinical immunotherapy investigations.