Isabelle Camby

In vitro influence of Phaseolus vulgaris, Griffonia simplicifolia, concanavalin A, wheat germ, and peanut agglutinins on HCT-15, LoVo, and SW837 human colorectal cancer cell growth.

BACKGROUND/AIMS: Compared with normal colonic mucosa, lectin receptor expression is increased in hyperplastic and neoplastic tissues; some lectins have been shown to influence human colonic epithelial cell proliferation. The aim was to assess further the influence of five lectins (Phaseolus vulgaris (PNA), Griffonia simplicifolia (GSA), concanavalin A (Con A), wheat germ (WGA), and peanut (PHA-L) agglutinins) on cellular growth in three human colorectal cancer cell lines (LoVo, HCT-15 and SW837). METHODS: Cells were cultured in four lectin concentrations (0.1, 1.0, 10, and 100 micrograms/ml) and growth assessed at days 2, 3, 5, and 7. The experiments were performed in media supplemented with either 1% or 10% fetal calf serum (FCS). Growth was assessed using the MTT (3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) colorimetric assay. RESULTS: Growth in each cell line was greatly affected by at least two of the lectins tested. There was some variation in the effect of a given lectin on different cell lines. Lectin effects showed a dose-response and the greatest effects generally resulted from the highest concentrations at the longest culture time. WGA and Con A induced large effects in all cell lines; the effects of Con A were partly blocked by the higher concentration of FCS. PNA had modest and uniform stimulatory effects overall. The effects of GSA and PHA-L varied between cell lines. CONCLUSIONS: The lectins studied all have the potential to affect colonic cancer growth in vitro. Many dietary lectins are resistant to digestion and may have important effects in vitro but the definition of their role in human colonic cancer biology must take into account the variability in lectin response.

Improving morphology-based malignancy grading schemes in astrocytic tumors by means of computer-assisted techniques.

We propose an original methodology which improves the accuracy of the prognostic values associated with conventional morphologically‐based classifications in supratentorial astrocytic tumors in the adult. This methodology may well help neuropathologists, who must determine the aggressiveness of astrocytic tumors on the basis of morphological criteria. The proposed methodology comprises two distinct steps, i.e. i) the production of descriptive quantitative variables (related to DNA ploidy level and morphonuclear aspects) by means of computer‐assisted microscopy and ii) data analysis based on an artificial intelligence‐related method, i.e. the decision tree approach. Three prognostic problems were considered on a series of 250 astrocytic tumors including 39 astrocytomas (AST), 47 anaplastic astrocytomas (ANA) and 164 glioblastomas (GBM) identified in accordance with the WHO classification. These three problems concern i) variations in the aggressiveness level of the high‐grade tumors (ANA and GBM), ii) the detection of the aggressive as opposed to the less aggressive low‐grade astrocytomas (AST), and iii) the detection of the aggressive as opposed to the less aggressive anaplastic astrocytomas (ANA). Our results show that the proposed computer‐aided methodology improves conventional prognosis based on conventional morphologically‐based classifications. In particular, this methodology enables some reference points to be established on the biological continuum according to the sequence AST→ANA→GBM.

DNA histogram typing in a series of 707 tumors of the central and peripheral nervous system.

The diagnostic value of ploidy level was investigated in a series of 707 tumors (from 707 patients) of the central and peripheral nervous system. The series contained 91 nerve sheath tumors (NSTs), 222 meningiomas (MNGs), 37 primitive neuroectodermal tumors (PNETs), 293 glial tumors of the adult (ATTs), and 64 brain metastases (MTTs). The ploidy level of each tumor was obtained by means of its DNA histogram type (DHT), which was computed (digital cell image analysis) on Feulgen-stained nuclei from archival formalin-fixed paraffin-embedded materials. The data reveal that the measurement of the ploidy level showed a diagnostic value in some of these tumor cases. Indeed, the proportion of highly aneuploid cases significantly (p < 0.05–0.01) decreased according to the sequence PNETs (72%) ± MTTs (64%) ± ATTs (40%) ± NSTs (23%) ± MNGs (9%). Nevertheless, this significant diagnostic value in terms of a distinction between benignity and malignancy only appears when all the tumors are taken into consideration. Indeed, at the level of one individual patient, our study shows that, like traumatic neuromas, schwannomas, or classical meningiomas, completely benign tumors can exhibit a high ane-uploidy level. In contrast, some tumors from histopathological groups like brain metastases or PNETs, which are known to be clinically aggressive, could be completely diploid.

Characterization of astroglial versus oligodendroglial phenotypes in glioblastomas by means of quantitative morphonuclear variables generated by computer-assisted microscopy

The current WHO classification places glioblastomas in the astrocytoma category. However, whether or not glioblastomas also show oligodendroglial differentiation remains a matter of controversy. This study investigates, at the morphonuclear level, the hypothesis that some glioblastomas (GBMs) may also represent the ultimate level of malignancy in the oligodendroglial lineage. Using a series of 164 GBMs, we sought to ascertain whether any of these GBMs exhibited phenotypical characteristics that were more closely related to oligodendroglial lineages than astrocytic lineages. Phenotypical features were quantitatively determined by means of the computer-assisted microscope analysis of Feulgen-stained nuclei, a process that made it possible to quantitatively describe the patterns of the cell nuclei (and, more specifically, of their chromatin) through 16 variables, and the distribution of the nuclear DNA content (DNA ploidy) through 8 variables. The phenotypical characteristics typical of astrocytic and oligodendroglial tumors were analyzed by means of Discriminant Analysis, a statistical multivariate analysis, performed on a series of 65 astrocytic and oligodendroglial tumors. This series consisted of 14 WHO grade II and 19 grade III astrocytomas and 24 WHO grade II and 8 grade III oligodendrogliomas. This multivariate analysis enabled an accurate model to be produced that distinguished between astrocytomas and oligodendrogliomas on the basis of 5 cytometry-generated variables. This model was used to characterize the phenotype of each of the 164 glioblastomas. The results show that of these 164 glioblastomas, 6 (about 3.5%) displayed phenotypes that were very similar to oligodendrogliomas, and 141 displayed phenotypes that were very similar,to astrocytomas. The phenotypes of the 17 remaining GBMs were too ambiguous to be categorized as having a pure astrocytic or oligodendroglial lineage.

Methodological aspects of using decision trees to characterise leiomyomatous tumors.

The aim of the present work is to present the potential uses of a classification technique labeled the decision tree for tumor characterisation when faced with a large number of features. The decision tree technique enables multifeature logical classification rules to be produced by determining discriminatory values for each feature selected. In this report, we propose a methodology that used decision trees to compare and evaluate the information contributed by different types of features for tumor characterisation. This methodology is able to produce a set of hypotheses related to a diagnosis and or prognosis problem. For example, hypotheses can be producted (on the basis of a set of descriptive features) to explain why tumor cases belong to a given histopathological group. To illustrate our purpose, this methodology was applied to the difficult problem of leiomyomatous tumour diagnosis. The aim was to illustrate what kind of diagnostic information can be extracted from a sample data set including 23 smooth muscle tumors (14 benign leiomyomas and 9 malignant leiomyosarcomas) described by a large set of computer-assisted, microscope-generated features. Three groups of features were used relating to: (1) ploidy level determination (10 features), (2) quantitative chromatin pattern description (15 features), and (3) immunohistochemically related antigen specificities (6 features). All these features were quantified by digital cell image analysis. The results suggest that an objective distinction between leiomyomas and leiomyosarcomas can be established by means of simple logical rules depending on only a few features among which the immunohistochemically revealed antigen expression of desmin plays a preponderant part. One of the combinations of features proposed by the methodology is interesting for pathologists, because it includes two features describing the appearance of a nucleus in terms of chromatin distribution homogeneity and density, two features widely used by pathologists in tumor-grading systems.

Identification of High Versus Lower Risk Clinical Subgroups in a Group of Adult Patients with Supratentorial Anaplastic Astrocytomas

The present work investigates whether computer-assisted techniques can contribute any significant information to the characterization of astrocylic tumor aggressiveness. Two complementary computer-assisted methods were used. The first method made use of the digital image analysis of Feulgen-stained nuclei, making it possible to compute 15 morphonuclear and 8 nuclear DNA content-related (ploidy level) parameters. The second method enabled the most discriminatory parameters to be determined. This second method is the Decision Tree technique, which forms part of the Supervised Learning Algorithms. These two techniques were applied to a series of 250 supratentorial astrocytic tumors of the adult. This series included 39 low-grade (astrocytomas, AST) and 211 high-grade (47 anaplastic astrocytomas, ANA, and 164 glioblastomas, GBM) astrocytic tumors. The results show that some AST, ANA and GBM did not fit within simple logical rules. These “complex” cases were labeled NC-AST, NC-ANA and NC-GBM because they were “non-classical” (NC) with respect to their cytological features. An analysis of survival data revealed that the patients with NC-GBM had the same survival period as patients with GBM. In sharp contrast, patients with ANA survived significantly longer than patients with NC-ANA. In fact, the patients with ANA had the same survival period as patients who died from AST, while the patients with NC-ANA had a survival period similar to those with GBM. All these data show that the computer-assisted techniques used in this study can actually provide the pathologist with significant information on the characterization of astrocytic tumor aggressiveness.

Quantitative chromatin pattern description in Feulgen-stained nuclei as a diagnostic tool to characterize the oligodendroglial and astroglial components in mixed oligo-astrocytomas.

The oligoastrocytoma, as a mixed glioma, represents a nosologic dilemma with respect to precisely defining the oligodendroglial and astroglial phenotypes that constitute the neoplastic cell lineages of these tumors. In this study, cell image analysis with Feulgen-stained nuclei was used to distinguish between oligodendroglial and astrocytic phenotypes in oligodendrogliomas and astrocytomas and then applied to mixed oligoastrocytomas. Quantitative features with respect to chromatin pattern (30 variables) and DNA ploidy (8 variables) were evaluated on Feulgen-stained nuclei in a series of 71 gliomas using computer-assisted microscopy. These included 32 oligodendrogliomas (OLG group: 24 grade II and 8 grade III tumors according to the WHO classification), 32 astrocytomas (AST group: 13 grade II and 19 grade III tumors), and 7 oligoastrocytomas (OLGAST group). Initially, image analysis with multivariate statistical analyses (Discriminant Analysis) could identify each glial tumor group. Highly significant statistical differences were obtained distinguishing the morphonuclear features of oligodendrogliomas from those of astrocytomas, regardless of their histological grade. When compared with the 7 mixed oligoastrocytomas under study, 5 exhibited DNA ploidy and chromatin pattern characteristics similar to grade II oligodendrogliomas, 1 to grade III oligodendrogilomas, and 1 to grade II astrocytomas. Using multifactorial statistical analyses (Discriminant Analysis combined with Principal Component Analysis), it was possible to quantify the proportion of “typical” glial cell phenotypes that compose grade II and III oligodendrogliomas and grade II and III astrocytomas in each mixed glioma. Cytometric image analysis may be an important adjunct to routine histopathology for the reproducible identification of neoplasms containing a mixture of oligodendroglial and astrocytic phenotypes.

Neurotensin-mediated effects on astrocytic tumor cell proliferation

Neurotensin (NT) and neurotensin receptors (NTRs) are widely found in the brain, NT may be considered as a mitogen factor in some tissues. However, no NT-mediated effects on glioma cell proliferation have been reported so far. In our present study we investigated the influence of NT on the proliferation of astrocytic tumor cell lines. To this end we used a synthetic NT agonist (JMV-449), a protease inhibitor which blocks the natural degradation of NT (JMV-531), and NT. The in vitro biological models used in the present study included the low grade SW1088, and the high grade U87, U373 and A172 astrocytic tumor cell lines. The peptide-induced influence on astrocytic tumor cell proliferation was investigated by means of the colorimetric MTT assay. Our results show that the NT and the NT agonist significantly stimulated the proliferation in 2/4 and 3/4 of the astrocytic cell lines respectively. Similarly, compound JMV-531 also induced an increase in the proliferation of 2/4 of the astrocytic cell lines. This marked influence of the NT and NT agonists, or the enzyme-endogenous prevention of its degradation on the regulation of astrocytic tumor growth therefore suggests that NT antagonists might be used to treat certain patients with high grade astrocytic tumors that do not respond to chemotherapy and/or radiotherapy.

Characterisation of the influence of anti-gastrin, anti-epidermal growth factor, anti-oestradiol, and anti-luteinising hormone releasing hormone antibodies on the proliferation of 27 cell lines from the gastrointestinal tract.

Numerous data from published reports prove that the proliferation of gastrointestinal tumour cell lines are under the control of many hormones or growth factors, or both. Most of these publications report the influence on a very small number of cell lines of one or two such factors only. This work deals with the in vitro characterisation of the influence of the anti-gastrin, the anti-epidermal growth factor (EGF), the anti-oestradiol (E2), and the anti-luteinising hormone releasing hormone (LHRH) antibodies on the proliferation of a large series of gastrointestinal cell lines. Cell proliferation was assessed by means of the colorimetric MTT assay on a series of 27 gastrointestinal cell lines obtained from the American Type Culture Collection (ATCC). Of the 27 cell lines, the anti-gastrin, the anti-EGF, the anti-E2, and the anti-LHRH neutralising antibodies considerably influenced the proliferation of 13, 25, 12, and 16. No gastrointestinal cell line was unresponsive to the four antibodies simultaneously. The anti-gastrin and anti-EGF antibody induced effects on the 27 gastrointestinal cell line proliferation were significantly correlated, as was also the case for the anti-E2 and anti-LHRH antibody induced effects. Of the anti-gastrin, the anti-EGF, the anti-E2, and the anti-LHRH antibodies, it was the anti-EGF one that had the greatest influence, both quantitatively and qualitatively, on gastrointestinal cell proliferation. The correlation of the effects of definite anti-hormone antibodies is suggestive of a common mechanism of action for the corresponding hormones and casts some doubt on the efficiency of anti-hormone monotherapy.

Assessment of nuclear size, nuclear DNA content and proliferation index in stereotaxic biopsies from brain tumours

In this paper we study the feasibility of measuring the diverse biological parameters in stereotaxic biopsies from human brain lesions. These biological parameters are the nuclear area (NA), the proliferation index (PI) and the ploidy level, the latter of which was evaluated by means of the DNA index (DI) and histogram type (DHT). These parameters were assessed by means of the digital cell image analysis of Feulgen‐stained nuclei. This analysis was performed on 124 samples from 22 computed tomography (CT)‐guided stereotaxic biopsies. The data show that the methodology used here enables the above parameters to be assessed on small samples without limiting the classical anatomopathological diagnosis. The data also reveal that the DHT corresponded more accurately to the ploidy level in the sample analysed than the DI. Lastly, it appears that supratentorial astrocytic tumours of the adult, which constituted the majority of the cases analysed here, are strongly heterogeneous at a biological level.