Systematic development of a mobile preconception lifestyle programme for couples undergoing IVF: the PreLiFe-programme.

STUDY QUESTION: Can we develop a preconception lifestyle programme for couples undergoing IVF that is in line with their needs. SUMMARY ANSWER: A mobile preconception lifestyle programme was systematically developed based on expert opinion, literature and needs of IVF-patients. WHAT IS KNOWN ALREADY: A healthy lifestyle prior to conception is not only beneficial for the general health of couples, but evidence on its importance for their reproductive health and the health of their children is also emerging. So far, the vast majority of fertility clinics do not offer a lifestyle programme for couples undergoing IVF. Therefore, the present study aimed to develop a lifestyle programme for IVF-couples. STUDY DESIGN, SIZE, DURATION: The development of the PreLiFe-programme was guided by the steps of the Medical Research Council (MRC) framework for developing complex interventions, a systematic approach for developing theory- and evidence-based health promotion interventions. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: First, the evidence base on lifestyle programmes for IVF-couples was reviewed. Second, several iterations between an expert panel, the literature, and quantitative and qualitative data from IVF-patients identified the content, the format, behaviour change techniques and theory of the programme. Third, the PreLiFe-programme was produced and the expected process and outcomes of a randomized controlled trial assessing it were modelled. Finally, user tests among experts and patients and pilot tests among patients were conducted. MAIN RESULTS AND ROLE OF CHANCE: The finally developed PreLiFe-programme is a mobile application to be used autonomously by both partners of IVF-couples during the first year of IVF, in combination with motivational interviewing over the telephone every three months (i.e. blended care). The PreLiFe-programme provides advice and skills training on physical activity, diet and mindfulness based stress reduction and is in part tailored based on monitoring and tracking the lifestyle of patients. Based on the literature the expert panel considers it plausible that all three components contribute to IVF-success rates. The PreLiFe-programme is likely to be acceptable to patients as it meets the need of patients for lifestyle advice and treatment information. LIMITATIONS, REASON FOR CAUTION: The pilot in IVF-couples had a 3-month duration. The feasibility of the PreLiFe-programme in other infertile populations and/or upon longer use is yet to be examined. Whether the PreLiFe-programme effectively improves lifestyle and IVF-success rates is currently being examined in a trial randomizing heterosexual couples starting IVF to the PreLiFe-programme or an attention-control group for 12 months. WIDER IMPLICATIONS OF THE FINDINGS: If the PreLiFe-programme improves lifestyle and the chance of IVF-success, it will be a powerful tool and provide guidance for implementing lifestyle programmes in fertility clinics. STUDY FUNDING/COMPETING INTEREST(S): Funded by the Research Foundation Flanders (FWO-TBM (Applied Biomedical Research with a Primary Social finality); reference: T005417N). The authors have no conflict of interest to report. TRIAL REGISTRATION NUMBER: NCT03790449.

Quantitative evaluation of single cell spread on collagen matrices.

Cells change their morphology as a response to environmental cues. The quantitative evaluation of single cell spread on extracellular matrices, such as type I collagen, is a key tool in cancer research. Inherent to the manual scoring of cellular spread is inter-observer but also intra-observer variation. To overcome these problems, we have developed the Morphology Analysis Software (MAS). MAS scores phase-contrast images of cells on native type I collagen gels and identifies whether a cell has a spread or round morphology using a combination of four unique parameters: the presence of a cellular extension, the cell area, the cell eccentricity and cell circularity. The MAS software scores are equivalent to the average score of five independent observers but MAS is faster, more objective and standardized. A functional screening assay using six cytokines identified TGFα as a stimulator of HCT8/E11 and SK-BR-3 single cell spreading on top of type I collagen gels. This change in morphology correlates with increased migration potential as evidenced by xCELLigence migration assays and are counteracted by EGFR signaling pathway inhibitors. This underscores the use of morphology classification on a population of unlabeled cells as read-out of an important cancer cell property and the potential for the MAS software in drug screening strategies.

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 assay's 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.

Determination of tumour prognosis based on angiogenesis-related vascular patterns measured by fractal and syntactic structure analysis.

AIMS: Intratumoural micro-vessel density (IMD) has recently been shown to be a valuable prognostic tool in many tumours. Yet, IMD does not take into account the spatial arrangement of the vessels, therefore only partly reflecting the angiogenic situation. In order to describe contextual vascular relationships more accurately, we have used fractal and syntactic structure analysis (SSA) based on computerised image processing to quantify micro-vascular hot spots. MATERIALS AND METHODS: The parametric performance in prediction of patients' outcome was evaluated by univariate analysis and compared with manually obtained IMDs, whereas an automated K-nearest-neighbour (KNN) classifier searched most discriminative parametric combinations. The method is based on analysis of vascular 'hot-spots' of paraffin-embedded tissue sections of invasive cervical carcinoma, colorectal carcinoma and malignant mesothelioma. RESULTS: For all three cancers, prediction of prognosis based on SSA yielded in general much higher recognition scores compared with IMD or fractal dimension. Survival of cervical carcinoma was mostly correlated with clinical data, with the vascular permeation being the only parameter with independent value. Prognosis of colorectal carcinoma is best described by SSA, completed with IMD, indicating an inverse correlation of survival time with a more irregular pattern and a slight increase in vessel number. For mesothelioma, we found a strong correlation with SSA and patients' outcome, with two SSA-parameters having independent prognostic value. CONCLUSIONS: The more accurate angiogenic description obtained with SSA may be useful for further exploitation as a prognosticator in a general diagnostic pathology service.

Transcontinental communication and quantitative digital histopathology via the Internet; with special reference to prostate neoplasia.

OBJECTIVE: To describe practical experiences in the sharing of very large digital data bases of histopathological imagery via the Internet, by investigators working in Europe, North America, and South America. MATERIALS: Experiences derived from medium power (sampling density 2.4 pixels/microm) and high power (6 pixels/microm) imagery of prostatic tissues, skin shave biopsies, breast lesions, endometrial sections, and colonic lesions. Most of the data included in this paper were from prostate. In particular, 1168 histological images of normal prostate, high grade prostatic intraepithelial neoplasia (PIN), and prostate cancer (PCa) were recorded, archived in an image format developed at the Optical Sciences Center (OSC), University of Arizona, and transmitted to Ancona, Italy, as JPEG (joint photographic experts group) files. Images were downloaded for review using the Internet application FTP (file transfer protocol). The images were then sent from Ancona to other laboratories for additional histopathological review and quantitative analyses. They were viewed using Adobe Photoshop, Paint Shop Pro, and Imaging for Windows. For karyometric analysis full resolution imagery was used, whereas histometric analyses were carried out on JPEG imagery also. RESULTS: The three applications of the telecommunication system were remote histopathological assessment, remote data acquisition, and selection of material. Typical data volumes for each project ranged from 120 megabytes to one gigabyte, and transmission times were usually less than one hour. There were only negligible transmission errors, and no problem in efficient communication, although real time communication was an exception, because of the time zone differences. As far as the remote histopathological assessment of the prostate was concerned, agreement between the pathologist's electronic diagnosis and the diagnostic label applied to the images by the recording scientist was present in 96.6% of instances. When these images were forwarded to two pathologists, the level of concordance with the reviewing pathologist who originally downloaded the files from Tucson was as high as 97.2% and 98.0%. Initial results of studies made by researchers belonging to our group but located in others laboratories showed the feasibility of making quantitative analysis on the same images. CONCLUSIONS: These experiences show that diagnostic teleconsultation and quantitative image analyses via the Internet are not only feasible, but practical, and allow a close collaboration between researchers widely separated by geographical distance and analytical resources.

Nuclear chromatin patterns in normal, hyperplastic and atypical endometrium.

OBJECTIVE: To characterize the nuclei of endometrial lesions for the diagnostic categories of normal glandular tissue, simple hyperplasia, atypical hyperplasia and adenocarcinoma of the endometrium, with the specific goal of probing for heterogeneity. STUDY DESIGN: For each diagnostic category the images of 360 nuclei were recorded on a high-resolution video microphotometer. Features descriptive of the statistical and spatial distribution of nuclear chromatin were computed for each nucleus. A nonsupervised learning algorithm, P-index, was employed to establish subsets of nuclei within each diagnostic category and to determine whether these subsets were statistically significantly different in the nuclear chromatin pattern. RESULTS: Lesions from cases of hyperplasia, atypical hyperplasia and adenocarcinoma of the endometrium each contained several subsets of nuclei with statistically significantly different chromatin patterns. For one such subset from each diagnostic category, a clear trend of progression toward adenocarcinoma could be demonstrated. CONCLUSION: The nuclei in endometrial lesions represent a highly heterogeneous set. Any measure of lesion progression or regression due to chemopreventive intervention, in an individual case, will have to examine the proportion of nuclei in each of these subsets as well as measures of deviation from normal for each subset.

Validation of nuclear texture, density, morphometry and tissue syntactic structure analysis as prognosticators of cervical carcinoma.

OBJECTIVE: To evaluate the performance of karyometry and histometry in the prediction of survival, recurrence and response of early-stage invasive cervical carcinoma. STUDY DESIGN: Nuclear morphometry, chromatin texture and tissue architecture (characterized by syntactic structure analysis) were measured using a semiautomated image analysis system on 46 cases of Feulgen-stained tissue sections. The performance of the features was compared to that of clinical features, reported to be the best prognosticators until now, such as age, lympho-vascular permeation, histologic type, stage and grade. A K nearest neighbor classifier was used for classification. RESULTS: In the prediction of three-year survival, recurrence and response, syntactic structure analysis proved to be the best performer. Classification rates were, respectively, 100%, 94.4% and 94.5%. In all classifications, karyometric and histometric features outperformed clinical features. In general, the best performing features described differences in second-order population statistics (standard deviations). CONCLUSION: The results show that a quantitative analysis based on nuclear morphology, chromatin texture and histology can be considered an excellent aid in the prognosis of invasive cervical carcinoma. The measurements are not hampered by the need to undertake complete resections and are suited to daily practice when implemented in a semiautomated image analysis system.

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.

Data representation and reduction for chromatin texture in nuclei from premalignant prostatic, esophageal, and colonic lesions.

BACKGROUND: To identify nuclei and lesions with great specificity, a large set of karyometric features is arranged in the form of a linear profile, called a nuclear signature. The karyometric feature values are normalized as z-values. Their ordering along the profile axis is arbitrary but consistent. The profile of the nuclear signature is distinctive; it can be characterized by a new set of variables called contour features. A number of data reduction methods are introduced and their performance is compared with that of the karyometric features in the classification of prostatic, colonic, and esophageal lesions. METHODS: Contour characteristics were reduced to descriptive statistics of the set of z-values in the nuclear signature and to sequence information. The contour features derived were (1) relative frequencies of occurrence of z-values and of their differences and (2) co-occurrence statistics, run lengths of z-values, and statistics of higher-order dependencies. Performance was evaluated by comparing classification scores of diagnostic groups. RESULTS: Rates for correct classification by karyometric features alone and contour features alone indicate equivalent performance. Classification by a combined set of features led to an increase in correct classification. CONCLUSIONS: Image analysis and subsequent data reduction of nuclear signatures of contour features is a novel method, providing quantitative information that may lead to an effective identification of nuclei and lesions.

Wavelets as chromatin texture descriptors for the automated identification of neoplastic nuclei.

Chromatin distribution reflects the organization of the DNA of a nucleus and contains important cellular diagnostic and prognostic information. Feulgen staining of breast tissue enables the chromatin distribution of the nucleus to be visualized in the form of texture. Describing texture in an objective and quantitative way by means of a set of texture parameters, combined with the study of the relationship of such parameters to the pathobiological cell properties, is useful both for reduction of the subjectivity inherently coupled to visual observation and for more accurate prognosis or diagnosis. We have presented an automated classification scheme for the diagnosis and grading of invasive breast cancer. The input to this scheme was a digitized microscopical image, from which nuclei were segmented. Chromatin texture was described using a set of textural parameters that include first- and second-order statistics of the image grey levels. The more recently developed wavelet energy parameters were also included in our study. Classification was performed by a Knn-classifier, which is a versatile multivariate statistical classification technique. We investigated the role of the tissue preparation technique and found that parameters derived from cytospins were better texture descriptors than those from sections. A 100% correct classification was achieved in a patient diagnosis experiment and 82% in a nuclear grading experiment.

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.

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.

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.

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.

Atomic force microscopy: influence of air drying and fixation on the morphology and viscoelasticity of cultured cells.

The influence of fixation, air-drying and liquid-imaging on the morphology as well as on the viscoelasticity of malignant mesothelioma cells was studied by atomic force microscopy. In this study, dehydrated cells were more easily scanned and offered faster data recording than hydrated cells. However, the influence of fixation strength was more noticeable. Strong fixation induced flattening of the cytoplasm and loss of nuclear structure, resulting in a clearly visible cytoskeleton which could be easily seen as fibres orientated in the direction of the cell growth. By contrast, the morphology of hydrated cells was influenced to a lesser degree on fixation and showed an overall 'rounding' of the surface with vague, ill-defined structures. Nuclear areas of these samples were difficult to image. Viscoelasticity measurements also exhibited large differences. Dehydrated cells were much harder and showed a uniform indentation profile over the whole cell that was independent of fixation. Indentation on hydrated cells was large and depended on the height of the measuring spot, the submembranous structure and, to a lesser extent, on fixation. To calculate an overall 'cellular' viscoelasticity, different methods were tested on these samples. Indentations of multiple, randomly chosen points, covering the whole cell, were measured and averaged to yield a mean indentation score. We avoided the thin and shadowed areas since it was shown that these regions were less suited for measuring. Using this design, large viscoelasticity differences were found, on which the influence of the external parameters could be shown. In another set-up, layered imaging was tried. However, long data acquisition times caused cellular activation and rearrangement, making this scanning mode unsatisfactory.

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.

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 semiquantitatively 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.

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.