Barbara Weyn

Syndecan‐1 expression in malignant mesothelioma: correlation with cell differentiation, WT1 expression, and clinical outcome

Syndecan‐1 binds basic fibroblast growth factor (bFGF), modulates neovascularization, plays a role in epithelial differentiation and is up‐regulated by WT1. Malignant mesothelioma of the pleura is one of the most aggressive tumours known and expresses high levels of angiogenic growth factors. This study has analysed syndecan‐1 expression in mesothelioma tumours and cell lines by immunohistochemistry and immunoblotting, using anti‐syndecan‐1 antibody directed against the core protein, and has examined its relation to morphology, bFGF, WT1, and intra‐tumoural microvascular density (IMD). Shedding of syndecan‐1 in the conditioned medium of mesothelioma cell lines was detected in variable amounts. These studies indicate that (1) there is no correlation of syndecan‐1 with either bFGF expression or IMD in mesotheliomas in vivo; (2) syndecan‐1 is strongly expressed in the epithelial type of mesothelioma and in the epithelial component of biphasic mesotheliomas and the expression is reduced or lost in sarcomatoid differentiation; together with the finding that (3) syndecan‐1 correlates with WT1 immuno‐expression, this suggests that syndecan‐1 might relate to the differentiation state of mesothelial/mesothelioma cells; and (4) syndecan‐1‐positive tumours are associated with a longer survival (p =0·02) than mesotheliomas with no or little syndecan‐1 expression, on univariate analysis. These findings therefore indicate that syndecan‐1 can be an important prognostic indicator in mesotheliomas and its loss may be important in the epithelial–mesenchymal transformation of mesothelioma cells. Copyright

Evaluation of tumour angiogenesis as a prognostic marker in malignant mesothelioma.

Angiogenesis plays an important role in the growth, progression, and metastasis of solid tumours. Malignant mesothelioma (MM) of the pleura is a highly invasive tumour with a poor prognosis. In the present study, microvascular quantification was undertaken on 25 specimens of mesothelioma and 15 specimens of non‐neoplastic mesothelium (NNM), by staining for the antigens CD34 and CD31. Areas of highest intratumoural microvascular density (IMD) were identified and counted either manually (mIMD) or on a computerized image analysis system (CIAS; iIMD). The two IMDs were significantly correlated with each other (r=0·736; P<0·001). The average IMD for MM was significantly (P<0·001) higher than in NNM. Moreover, each unit increment in iIMD for MM, when regarded as a continuous variable, was significantly (P=0·001) associated with an increased hazard of about 4 per cent. When regarded as a categorical variable, the patients in the highest tertile (>58 vessels/field) had a significantly (P<0·01; log‐rank test) shorter survival than patients in the lowest tertile (<45 vessels/field). This association was independent of the age of the patient and of the histological type or grade of the MM. No association was noted with p53 immunoexpression. Although the mean vascular area of blood vessels measured on the CIAS did not correlate with survival, assessment of IMDs can be an important independent prognostic indicator in malignant mesothelioma.

Automated breast tumor diagnosis and grading based on wavelet chromatin texture description

In this paper, wavelets were employed for multi-scale image analysis to extract parameters for the description of chromatin texture in the cytological diagnosis and grading of invasive breast cancer. Their value was estimated by comparing the performance of co-occurrence, densitometric, and morphometric parameters in an automated K-nearest neighbor (Knn) classification scheme based on light microscopic images of isolated nuclei of paraffin-embedded tissue. This design allowed a multifaceted cytological retrospective study of which the practical value can be judged easily. Results show that wavelets perform excellently with classification scores comparable with densitometric and co-occurrence features. Moreover, because wavelets showed a high additive value with the other textural groups, this panel allowed a very profound description with higher recognition scores than previously reported (76% for individual nuclei, 100% for cases). Morphometric parameters performed less well and only slightly increased correct classification. The major drawback, besides image segmentation errors demanding operator supervision, emanated to be the few false-negative cases, which restrict the immediate practical use. However, an enlargement of the parameter set may avoid this misclassification, resulting in an applicable expert system of practical use.

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, Gabriels 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.

Expression profile of telomerase subunits in human pleural mesothelioma.

Using the TRAP assay, telomerase activity was previously detected in over 90% of human pleural mesotheliomas (MMs), but not in mesothelial cell cultures (MCCs), suggesting that telomerase re‐activation occurs during multi‐step mesothelioma carcinogenesis. The present study determined the expression of the telomerase RNA template (hTERC), the telomerase‐associated protein (hTEP1), and the telomerase catalytic sub‐unit (hTERT), in 16 pleural MMs and 4 MM‐derived cell lines, in two pleural solitary fibrous tumours and in six MCCs. Reverse transcription‐polymerase chain reaction analysis revealed that hTERT mRNA expression parallels the activity status documented by the TRAP assay, whereas hTERC and hTEP1 mRNA are commonly expressed in all malignant and non‐malignant serosal cells and tissues. Three alternatively spliced hTERT transcripts were detected in all telomerase‐positive samples, whereas neither variant could be detected in the MCCs. Detection of the hTERT protein with a commercially available antibody was not successful. These results indicate that hTERT expression is rate‐limiting for human telomerase activity and that re‐activation, rather than up‐regulation, of hTERT expression can play a critical role in MM carcinogenesis. While waiting suitable anti‐hTERT antibodies, these results provide information for the design of hTERT mRNA‐specific in situ probes to study telomerase in archived pre‐malignant serosal lesions. Copyright

The association between vascular endothelial growth factor, microvessel density and clinicopathological features in invasive cervical cancer

OBJECTIVE The aim of this study was to analyse the vascular endothelial growth factor (VEGF) expression in a series of cervical carcinomas and to compare the results with the microvessel density (MVD) and clinicopathological features. STUDY DESIGN The immunoreactivity for VEGF was studied in 130 invasive cervical carcinomas and in 22 patients with a carcinoma in situ of the cervix. The results were compared with the MVD. RESULTS Staining for VEGF of less then 50% per slide occurred in 80% of the invasive carcinomas and in 82% of the in situ carcinomas. The median MVD was 261 vv/mm(2) (range: 11-1000) in the invasive group and 146 vv/mm(2) (range: 25-536) in the in situ group. Unlike the microvessel density there was no association between VEGF expression and survival. The MVD was higher in VEGF poorer (<50%) tumours (P=0.055). Beside tumour histology (P=0.012) there were no other significant relationships between the remaining histopathological findings and VEGF expression. CONCLUSION Tissue VEGF expression has no prognostic value in contrast with the MVD in patients with invasive cervical cancer.

Automated analysis of contractility in the embryonic stem cell test, a novel approach to assess embryotoxicity

The embryonic stem cell test (EST) is an ECVAM-validated assay to detect embryotoxicity. The output of the assay is the effect of test compounds on the differentiation of murine-derived embryonic stem cells (D3 cells), recorded by visual analysis of contracting cardiomyocyte-like cells. Incorporation of a system to assess the contractility in an automated manner is proposed, to increase the throughput in the EST independent of observer bias. The automated system is based on image recording of each well, resulting in the area (pixels) and frequency of contractility (Hz). Four test compounds were assessed for their embryotoxic potency in the 96-well version of the EST, with both manual and automated analysis: 6-Aminonicotinamide, Valproic Acid, Boric Acid, and Penicillin G. There was no statistically significant difference in the outcome of both methods in the fraction of contractility (p<0.05), resulting in the same rank-order of Relative Embryotoxic Potency (REP) values: 6-aminonicotinamide (1)>valproic acid (0.007-0.013)>Boric Acid (0.002-0.005)>Penicillin G (0.00001). The automated image recording of contractile cardiomyocyte-like cells in the EST allows for an unbiased high throughput method to assess the embryotoxic potency of test compounds, resulting in an outcome comparable to manual analysis.

Value of morphometry, texture analysis, densitometry, and histometry in the differential diagnosis and prognosis of malignant mesothelioma

Malignant mesothelioma is a tumour with increasing incidence due to widespread use of its causative agent, asbestos, in the past decades. The poor survival necessitates a correct differentiation from other lesions at the same site, such as hyperplastic mesothelium and carcinomas metastatic to pleura or peritoneum. Since genetic and immunohistochemical markers are not absolutely differentiating, the diagnosis is based on the histology complemented with (immuno)histochemistry. However, as the tumour presents itself in numerous heterogeneous histological forms, visual evaluation is extremely difficult. In order to evaluate the prognostic and diagnostic performance of syntactic structure analysis (SSA), chromatin texture analysis, densitometry, and morphometry, an automated KNN‐classification system has been used to compare Feulgen‐stained tissue sections of hyperplastic mesothelium, malignant mesothelioma, and pulmonary adenocarcinoma. In addition, we also studied most discriminative aspects in the differentiation, typing, and prediction of survival. The results indicate that for the diagnosis of malignant mesothelioma, chromatin texture parameters outperform SSA, densitometry, and morphometry (recognition score=96·8 per cent). Most discriminative parameters highlight spatial patterns of the chromatin distribution that are hard to appraise visually and directly show the benefits of a quantitative approach. Typing of the tumour is best described by SSA parameters, relating to the spatial arrangement of the cells in the tissue (recognition score=94·9 per cent). In survival time classifications, chromatin texture yields the highest recognition score (82·9 per cent), although accurate estimations are unreliable due to a large degree of misclassification. Copyright

Telomerase activity in human pleural mesothelioma

BACKGROUND Gradual telomere erosion eventually limits the replicative life span of somatic cells and is regarded as an ultimate tumour suppressor mechanism, eliminating cells that have accumulated genetic alterations. Telomerase, which has been found in over 85% of human cancers, elongates telomeres and may be required for tumorigenesis by the process of immortalisation. Malignant mesothelioma is an incurable malignancy with a poor prognosis. The disease becomes symptomatic decades after exposure to carcinogenic asbestos fibres, suggesting the long term survival of pre-malignant cell clones. This study investigated the presence of telomerase in pleural malignant mesothelioma, which may be the target for future anti-telomerase drugs. METHODS Telomerase activity was semi-quantitatively measured in extracts from 22 primary pleural mesotheliomas, two benign solitary fibrous tumours of the pleura, four mesothelioma cell lines, and six short term mesothelial cell cultures from normal pleura using a non-isotopic dilution assay of the telomeric repeat amplification protocol. RESULTS Twenty of the 22 primary mesotheliomas (91%) and all tumour derived mesothelioma cell lines were telomerase positive. Different levels of enzyme activity were observed in the tumours of different histological subtypes. Telomerase activity could not be detected in the six normal mesothelial cell cultures or in the two mesotheliomas. Both benign solitary fibrous tumours showed strong telomerase activity. CONCLUSIONS Telomerase activity is found in a high proportion of mesotheliomas and anti-telomerase drugs might therefore be useful clinically. The results are consistent with the hypothesis that telomerase activity may be a feature of carcinogenesis in mesotheliomas and possibly in many other cancers.

A multiparametric assay for quantitative nerve regeneration evaluation

We introduce an assay for the semi‐automated quantification of nerve regeneration by image analysis. Digital images of histological sections of regenerated nerves are recorded using an automated inverted microscope and merged into high‐resolution mosaic images representing the entire nerve. These are analysed by a dedicated image‐processing package that computes nerve‐specific features (e.g. nerve area, fibre count, myelinated area) and fibre‐specific features (area, perimeter, myelin sheet thickness). The assays performance and correlation of the automatically computed data with visually obtained data are determined on a set of 140 semithin sections from the distal part of a rat tibial nerve from four different experimental treatment groups (control, sham, sutured, cut) taken at seven different time points after surgery. Results show a high correlation between the manually and automatically derived data, and a high discriminative power towards treatment. Extra value is added by the large feature set. In conclusion, the assay is fast and offers data that currently can be obtained only by a combination of laborious and time‐consuming tests.