André Van Daele

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.

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.

Biochemical and histopathological changes in nephrectomized mice

Renal failure is characterized by the retention of nitrogenous metabolites such as urea, creatinine (CTN) and other guanidino compounds (GCs), uric acid, and hippuric acid, which could be related to the clinical syndrome associated with renal insufficiency. A model of renal failure has been developed in male C57BL x Swiss-Webster mice using nephrectomy (NX) and/or arterial ligation. A sham group (group A) and two nephrectomized groups, group B (one kidney removed) and group C (one kidney removed and ligation of the contralateral anterior artery branch), were studied. Ten days postsurgery, morphological and functional indices of renal failure were investigated. Nephrectomized mice manifested features of renal failure like polyuria and wasting. CTN clearance (CTN[Cl]) decreased by +/-26% in group B and +/-33% in group C as compared with the control values. Marked increases in the plasma concentration of guanidinosuccinic acid ([GSA] fourfold) and guanidine ([G] twofold) were observed in the experimental animals. CTN and alpha-keto-delta-guanidinovaleric acid (alpha-keto-delta-GVA) reached levels of, respectively, 1.5-fold and twofold those of controls. Urinary GSA excretion increased and guanidinoacetic acid (GAA) excretion decreased about twofold in group C. GSA increases (2.6-fold) were also observed in the brain in group C, in addition to a significant increase of G (2.5-fold) and gamma-guanidinobutyric acid ([GBA] 1.5-fold). Finally, the extent of NX was found to be 45.2% in group B and 71.4% in group C. Light microscopy revealed an expansion and increase in cellularity of the mesangium of the glomeruli, particularly in group C. A significant correlation (r = .574, P < .0001) was found between CTN(Cl) and the degree of NX as calculated from the remaining functional area. These data suggest that the model can be used as a tool for further pathophysiological and/or behavioral investigations of renal failure.

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

Angiogenesis in cervical intraepithelial neoplasia and the risk of recurrence.

OBJECTIVE We sought to investigate whether angiogenesis can predict the risk of recurrence of cervical intraepithelial neoplasia after treatment. STUDY DESIGN Microvessel density was studied in 75 patients with grade 3 cervical intraepithelial neoplasia and in 20 patients with microinvasive squamous carcinoma (International Federation of Gynecology and Obstetrics stage IA1) of the uterine cervix by staining representative tissue sections with the specific endothelial marker anti-CD31. The microvessel density was determined with a digital image analyzer. The results were correlated with clinical and histopathologic data. RESULTS The mean vessel density was 264 per field (range, 86-674 per field) in grade 3 cervical intraepithelial neoplasia and 378 per field (range, 161-848 per field; P = .001) in microinvasive squamous carcinoma. Thirteen patients with grade 3 cervical intraepithelial neoplasia had recurrent cervical intraepithelial neoplasia (microvessel density, recurrent vs nonrecurrent; not significant). Multiple regression analysis in the noninvasive group confirmed that the mean vessel density (P = .121) had no prognostic value. Furthermore, it showed that the age at diagnosis (P = .011), menopausal status (P = .052), and treatment modality (P = .022) proved to be independent prognostic factors for recurrence. CONCLUSIONS During the progression from noninvasive to microinvasive cervical carcinoma, the microvessel density increases significantly. However, the vessel density does not predict recurrence of noninvasive lesions.

Combined characterization of composite tabular silver halide microcrystals by cryo‐EFTEM/EELS and cryo‐STEM/EDX techniques

The combination of cryo‐energy filtering transmission electron microscopy (EFTEM)/electron spectroscopic diffraction (ESD)/electron energy‐loss spectroscopy (EELS) and cryo‐energy‐dispersive X‐ray (EDX) analysis in the scanning transmission (STEM) and scanning (SEM) modes was applied for the characterization of composite tabular Ag(Br,I) microcrystals. A low‐loss fine structure in EEL spectra between 4 and 26 eV was attributed to excitons and plasmons possibly superimposed with interband transitions and many‐electron effects. The contrast tuning under the energy‐filtering in the low‐loss region was used to image the crystal morphology, defect structure (random dislocations and {111―} stacking faults) and bend and edge contours as well as electron excitations in the microcrystals. Sharp extra reflections at commensurate positions in between the main Bragg reflections and diffuse honeycomb contours in ESD patterns of the microcrystals taken near the [111] zone were assigned to the number of defects in the shell region parallel to the grain edges and polyhedral clusters of interstitial silver cations, respectively. The imaginary part of the energy‐loss function, Im (‐1/ϵ), and the real and imaginary parts, ϵ1and ϵ2, of the dielectric permittivity were determined by means of a Kramers‐Kronig analysis. An assignment of exciton peaks based on calculations of electronic band structure of silver bromide is proposed. Inner‐shell excitation bands of silver halide were detected in line with EDX‐analyses. The energy‐loss near‐edge structure (ELNES) of the AgM4,5‐edge governed by spin‐orbital splitting between the 3d3/2‐ and 3d5/2‐states has been evaluated. Combined silver and halide distributions were obtained by a three‐window method (EFTEM) and by EDX/STEM including area mapping and line profiling of iodide. Microsc. Res. Tech. 42:108–122, 1998.

Fractal Dimension, Form and Shape Factors for the Quantification of Nuclear Signature Profiles

Patient-oriented diagnosis based on an accurate quantification of the lesion may improve the patient’s diagnosis/prognosis as compared to diagnosis/prognosis based on population statistics. A nuclear signature is a technique developed to quantify lesions with great specificity and accuracy and is an important tool for patient-oriented diagnosis. However, nuclear signatures are thus far mainly applied in visual evaluations. This paper describes a method to quantify nuclear signatures based on a fractal and geometrical analysis. The result - a new set of fractal-geometrical features - is evaluated by comparing its diagnostic performance with classical karyometric features on 3 tumour types. Results show that the diagnostic performance of the feature sets is similar but that classification scores are increased by combining the two sets. Moreover, classification rates of malignancy associated changes of colon lesions are higher by using the fractal-geometrical feature set, proving its ability to detect sub visual changes of early premalignant transformation.

Structural analysis of silver halide cubic microcrystals with epitaxial or conversion growths by STEM-EDX

Elemental distributions and contents of silver halide cubic microcrystals with epitaxial or conversion growths were analyzed by back scattered electron imaging, X-ray mapping and X-ray analysis in the scanning transmission electron microscope (STEM) combined with energy dispersive X-ray analysis (EDX). A liquid nitrogen cryostage was used to minimize sample damage and drift during electron bombardment. The quality of backscattered electron imaging was improved by selecting the optimal parameters of the image processing system and by off-line image processing. The back scattered electron images and X-ray maps show the elemental distribution in individual microcrystals. X-ray analyses in the spot mode yield semiquantitative results. Although the cubic shape of the microcrystals and their distorted appearance after tilting the sample make them difficult to be analyzed, this work indicates that the combination of these methods can be used to determine the elemental distribution and content in cubic microcrystals and that the analyses provide a valuable advice for the choice of the preparation conditions.