Ploidy and chromatin pattern analysis as an aid for cervical smear diagnosis.

In the present study we used computer-assisted microscopy to analyze the morphology of Feulgen-stained cell nuclei in cell populations obtained at the same time as routinely performed cervical smears and in the same way. We investigated in a series of 110 cases whether the quantitative morphonuclear description of cytological cervical samples is able to aid pathologists to distinguish between benign and more suspect premalignant lesions. For this task nuclear DNA content, nuclear morphometry (size and anisonucleosis level) and chromatin pattern-related parameters were compiled for each specimen enrolled in the database. A set of 32 normal and 17 high-grade squamous intraepithelial lesion (HSIL) specimens (with diagnostic confirmations) were selected as references and used to establish a discriminant model on the basis of cytometry-generated variables. This model was then used to score the remaining 61 cases in our series (including cases exhibiting benign cellular changes, squamous cells of undetermined significance, low-grade SIL and cancers). The results show that a model discriminating efficiently between normal and HSIL groups can be obtained by combining 5 quantitative features (1 DNA ploidy-related, 2 morphometrical and 2 chromatin texture features). A 97% specificity and an 88% sensitivity characterized the boundary so established. When applied to new cases, the model was in fact able to correct diagnoses for cases which had been down- or up-graded on the basis of the Bethesda system, and provided scores in accordance with histological control.

Galectin fingerprinting in tumor diagnosis. Differential expression of galectin-3 and galectin-3 binding sites, but not galectin-1, in benign vs malignant uterine smooth muscle tumors.

Cell-matrix interactions are governed by a distinct set of proteins, with 2 nonintegrin laminin-binding proteins, galectin-1 and galectin-3, providing 1 aspect. The expression patterns of laminin and the 2 galectins and galectin binding sites were quantitatively determined by means of computer-assisted microscopy with the aim of differentiating between 16 leiomyomas and 10 leiomyosarcomas of the uterus. Three quantitative variables were computed for each of the 5 histochemical markers: labeling index, which describes the percentage of tissue area specifically stained by a given marker; mean optical density which reflects the concentration of the marker; and concentrational heterogeneity, which characterizes the degree of heterogeneity of the marker distribution in the tumor tissue areas. The results reveal evident differences in the galectin-3-related parameters in the 2 tumors groups. Whereas the concentration of galectin-3 binding sites was significantly (P = .01) weaker in the leiomyosarcomas than in the leiomyomas, the percentages of tumor tissue expressing galectin-3 (P = .02) and its binding sites (P = .002) were significantly higher in the leiomyosarcomas than in the leiomyomas. Although significantly (P = .02) higher, the concentration of laminin was more heterogeneously distributed (P = .01) in the leiomyosarcomas than in the leiomyomas. In contrast, the levels of expression of galectin-1 and its accessible binding sites remained similar for both the leiomyomas and the leiomyosarcomas. Finally we document how the levels of expression of galectin-3 and its binding sites can be of assistance in reliably differentiating leiomyomas from leiomyosarcomas.

Growth inhibition of human in vitro and mouse in vitro and in vivo mammary tumor models by retinoids in comparison with tamoxifen and the RU-486 anti-progestagen.

Retinoids constitute a very promising class of agents for the chemoprevention or treatment of breast cancer. These retinoids exert their biological activity through two distinct classes of retinoic acid (RA) receptors (R), the RAR isotypes (alpha, beta, and gamma) and the three RXR isotypes (alpha, beta, and gamma) and their numerous isoforms which bind as RXR/RAR heterodimers to the polymorphic cis-acting response elements of RA target genes. With respect to these numerous receptor sub-types, the retinoid-induced effects at the biological level include marked modifications with respect to both cell proliferation and cell death (apoptosis), and also in the induction of differentiation processes. The present study aims to characterize the effect which four retinoids (TTNPB, 9-cis-RA, LGD 1069, 4-HPR) with distinct RAR/RXR binding properties induced on various in vitro and in vivo mouse and human breast cancer models. The experiments with the retinoids were carried out in comparison with the anti-estrogen tamoxifen and the anti-progestagen RU-486 compounds. The results show that the 6 compounds under study were markedly more efficient in terms of growth inhibition in the human T-47D cell line when maintained under anchorage-independent culture conditions than when maintained under anchorage-dependent ones. While RU-486 exhibited a weak statistically significant (p < 0.05) influence on the growth of the T-47D stem cells, tamoxifen had a marked inhibitory influence on the growth of these cells. Of the four retinoids, 4-HPR was the least effective since the lowest doses tested (1 and 0.1 nM) exhibited no statistically (p > 0.05) significant influence on the growth of the stem cells. The most efficient retinoid was TTNPB. It was only at the highest dose (10 microM) that tamoxifen and RU-486 showed a weak inhibitory influence on the growth of the T-47D non-stem cells while all 4 retinoids exerted a significant inhibitory influence on the growth of these non-stem cells, with 4-HPR being the most efficient (P < 0.001) at the highest dose, but ineffective (P > 0.05) at the lowest. Tamoxifen and TTNPB were tested in vivo on hormone-sensitive (HS) and hormone-insensitive (HI) strains of the MXT murine mammary carcinoma. While TTNPB appeared to be equally efficient in terms of growth inhibition in both MXT-HS and MXT-HI models, tamoxifen had only a marginal inhibitory influence on the growth of the MXT-HI strain but did inhibit growth in the case of the MXT-HS one. TTNPB was markedly more efficient than tamoxifen in terms of both inhibiting the cell proliferation level (measured by means of computer-assisted microscopy applied to Feulgen-stained nuclei, a method which enables the percentage of cells in the S phase of the cell cycle to be determined) and triggering cell death (measured by means of the determination of the transglutaminase activity) in both the MXT-HI and MXT-HS models. The very significant TTNPB-induced inhibition of the macroscopic MXT-HS growth rate relates to the triggering of cell death (apoptosis) rather than to an inhibition of cell proliferation. All these results clearly indicate that retinoids are very efficient agents against breast cancer, at least as efficient as tamoxifen.

Ploidy level determination and quantitative chromatin pattern description in pregnancy-associated breast cancers.

The present study deals with the characterization of hormone-sensitivity in pregnancy-associated breast cancers (PBCs). This characterization was carried out in 22 PBCs as opposed to 88 non-pregnancy-associated breast cancers (NPBCs). For this study, we used the digital cell image analysis of Feulgen-stained nuclei to assess the type of hormone-sensitivity. In a previous study it was demonstrated that the chromatin pattern in breast cancers is related to the amounts of estrogen receptors they contain. Our results demonstrated that the quantitative description of the chromatin pattern by means of 15 parameters (relating to morphometric, densitometric, and textural features) made it possible to identify typical cell nuclei populations in the PBC and NPBC groups. The use of specific statistical analyses (principal-components and discriminant) made it possible to quantify the proportion of each cell nucleus type in the PBCs. Furthermore, of the 22 PBCs under study, 13 contained a large majority of cell nuclei whose chromatin pattern was characteristic of hormone-sensitive cells, while 5 cases contained a large majority of typically hormone-insensitive ones. The remaining 4 cases contained a relatively similar proportion of typically hormone-sensitive and insensitive cell nuclei. The quantitative chromatin pattern description thus made it possible to characterize the hormone-sensitivity level in PBCs, whereas DNA ploidy level determination did not enable any such characterization to be carried out. The chromatin pattern assay described here, which enables hormone-sensitive pregnancy-associated breast cancers to be identified from hormone-insensitive ones independently from biochemical assays, should help the physician regarding therapy adaptation.

Characterization of chemotherapy-induced morphonuclear modifications in the P388 leukaemia and the MXT mammary tumour models of the mouse.

Chemotherapy-induced morphonuclear modifications were monitored in vivo by means of the digital cell image analysis of Feulgen-stained nuclei. Two experimental models were used, i.e. the P388 mouse leukaemia and the MXT mouse mammary carcinoma. The drugs used were doxorubicin, etoposide and cyclophosphamide. The results indicate that the chemotherapy induced a significant decrease in the MXT tumour growth and a significant increase in the survival of the P388 leukaemic mice. These effects were accompanied at the morphonuclear level by an increase in the nuclear area, by modifications in the DNA content in accordance with the effects of the drugs on the cell cycle and by several modifications in the chromatin texture in accordance with the model or the drugs studied. While there were neither homogeneous morphonuclear changes in all treatment groups nor clearcut correlations between the morphonuclear changes and tumour growth or the survival of the animals, the present study nevertheless shows that it is possible, at least partly, to monitor in vivo certain chemotherapy-induced effects occurring at the morphonuclear level, and subsequently to obtain information on the mode of action of the drugs.

Introduction of the cytological grading, the nuclear area, the DNA index and the DNA histogram type in the setting up of a score for ductal breast carcinoma.

The present study describes the setting up of a new score which makes it possible objectively to grade ductal breast carcinomas, i.e. not-otherwise-specified (NOS) cancers, on cellular material from fine-needle aspirations (FNAs). For this purpose FNAs from 252 patients--with NOS breast cancers--were smeared onto histological slides, fixed in an ethanol-formolacetic acid mixture, Feulgen-stained and analysed by means of a cell image processor. Four parameters were taken into account in setting up the score, namely the cytological prognostic grade (CPM) of malignancies similar to the Scarff-Bloom-Richardson (SBR) grading, the nuclear area (NA), the DNA index (DI) and the DNA histogram type (DHT). Each of these four parameters was considered as a "sub-score" which may take three values, i.e. 1, 2 and 3. The final result may thus range from 4 to 12. Subscores of 4 and 5 correspond to a cytological score of I, subscores of 6, 7 and 8 to a cytological score of II, sub-scores of 9 and 10 to a cytological score of III, and sub-scores of 11 and 12 to a cytological score of IV. In the present study, the results show 17% of CPM grade 1.52% of CPM grade II and 31% of CPM grade III cancers. All the cases exhibiting a cytological score of IV (5%) fully fit in with the CPM grade III cancers. In the same way, none of the cases exhibiting a score of I fit in with CPM grade III cancers. The cancers with a CPM grade II fit in with the scores of II and III. It thus seems possible to convert a three-value malignancy grading system (CPM and/or SBR grading) into a four-value one (cytological score). The main advantage in this latter type of system is that it becomes possible to split up the over-large group of CPM grade II cancers. As things stand, we are unable to give any prognostic value for the score proposed here because our study is prospective only. A study of this type has been necessary so as to provide against problems connected with ways of preserving specimens that might be used in a retrospective study. The bank of clinical and biological data now in existence must be allowed to mature for a number of years before the prognostic worth of the cytological score can be established, always assuming that such a value exists.

Digital cell image analysis of Feulgen-stained nuclei from human papillary, medullary, colloid, lobular and comedocarcinomas of the breast.

The morphonuclear characteristics (nuclear size and chromatin pattern), the proliferation index and the ploidy level were characterized in a series of 46 breast tumors including medullary (5 cases), papillary (6 cases), lobular (27 cases), colloid (4 cases) and comedo- (4 cases) carcinomas. The quantitative assessments were carried out by means of digital cell image analyses of Feulgen-stained nuclei from imprint smears. The results show that monovariate analyses (one-way variance analyses) were much less potent than multivariate analyses (principal components analyses followed by the canonical transformation of the data and discriminant analyses) in assessing the morphonuclear characteristics of these breast tumors. The multivariate analyses indicated that there might be a level of malignancy which increases according to the sequence papillary and medullary and colloid carcinomas-->comedocarcinomas-->lobular carcinomas. This assertion is corroborated by the ploidy-level-related results which revealed a higher proportion of highly aneuploid cases in the group of lobular carcinomas than in the group which included medullary, papillary and colloid carcinomas. However, since highly aneuploid cases were also encountered in this latter low malignancy level group, we expressed the hypothesis firstly that aneuploidy reflects two distinct biological properties, i.e. the aggressiveness of a tumor and its age, and secondly that a highly aneuploid but low malignancy tumor should correspond to old degenerating tumors.

DNA histogram typing and DNA index measurements in 508 invasive breast carcinomas from fine-needle aspirates and imprint smears as opposed to formalin-fixed paraffin-embedded tissues.

Morphometric, i.e. nuclear area (NA), and densitometric, i.e. nuclear DNA content, features were characterized in a series of 508 invasive ductal breast carcinomas. The specimens analyzed were from three distinct sources, i.e. fresh material (252 fine-needle aspirates as opposed to 147 imprint smears) immediately fixed in EFA fixative as opposed to archive material, i.e. 109 formalin-fixed paraffin-embedded tumours that were subsequently deparaffinized. Morphonuclear parameters were computed on Feulgen-stained nuclei by means of a cell image processor. Our results show that the development of nuclear size and DNA content in function of anatomopathological grading is approximately the same for specimens of breast cancer provided by FNAs, imprint smears and formalin-fixed paraffin-embedded tissues. However, in these latter instances it seems that part of the morphometric information is slightly modified in relation to the information obtained from fresh material directly fixed in EFA for cytophotometric analysis. The greatest discriminatory power of the morphometric parameters was obtained in relation to the FNAs. Lastly, in the present study we come out in favor of the idea that henceforth it would be preferable to express results concerning nuclear DNA content as DNA histogram types rather than in terms of DNA indices.

DNA histogram typing in retinoblastomas and neuroblastomas.

The nuclear DNA content characterization was carried out by means of both DNA index and DNA histogram type assessments in a series of 21 retinoblastomas, 11 neuroblastomas, 1 ganglioneuroblastoma, and 4 medulloblastomas. These measurements were performed by means of the cytophotometric digital cell image analyses of Feulgen-stained nuclei. The results indicate that as far as nuclear DNA content is concerned, retinoblastomas seem to be very different from neuroblastomas. In fact, in terms of DNA index, retinoblastomas are significantly more aneuploid than neuroblastomas. The DNA histogram type shows that the high level of aneuploidy found in retinoblastomas corresponds to genotypically polymorphic tumors, and this could reflect a serious degeneration of the genomic material in retinoblastomas. This type of degeneration seems to be much less frequent in neuroblastomas, which basically seem to be either diploid or hypertriploid.

Prevention of ovarian damage induced by cyclophosphamide in adult female mice by hormonal manipulations.

Doses of 10 or 20 mg cyclophosphamide/kg body weight were administered daily to mice for up to 20 days. This caused significant reductions in the incidence of prenatal (developing) follicles and significant increases in atretic (degenerating) follicles within the ovaries. Attempts to prevent cyclophosphamide-induced damage by simultaneous treatment with oestrogen alone, oestrogen plus progesterone, or danazol (a synthetic androgen) demonstrated that danazol effectively prevented the ovarian damage. The efficacy of danazol was considered to be due to its ability to inhibit LH/FSH secretion and, indirectly, the development of new ovarian follicles.

Effects of aminoglutethimide plus hydrocortisone on the genital tract of postmenopausal women with advanced breast cancer. A clinical and cytologic survey.

In this study we compared the effects of either Aminoglutethimide (AGL) or Tamoxifen (TAM) therapy on the genital tract of postmenopausal women with advanced breast cancer. Thus, 15 patients treated with AGL, and 10 patients treated with TAM underwent gynaecological examination, during which vaginal smears were taken. All smears were reviewed in blind by one pathologist for determination of karyopycnotic indices (KI). Under TAM, no significant clinical abnormality was observed, except in one patient who had a small (histologically benign) endocervical polyp, easily removed during the examination. As reported by others, smears made under TAM therapy were generally characterized by high KI, indicating that an hormonal, estrogen-like stimulation remained present in these patients. On the contrary, most women under AGL had some evidence of vulvovaginal atrophy, which was unvariably associated with low KI on smears. Among the latter, four had severe dystrophic lesions consisting of leukoplasia (1), kraurosis (2) or lichen sclerous and atrophicus (1). It is therefore recommended not to neglect the systematic practice of gynaecological examination in patients with advanced breast cancer under endocrine therapy. These observations also indicate that AGL and TAM exert entirely opposite effects on the vaginal mucosa, which is a very sensitive estrogen-target tissue. In good agreement with former endocrine studies, AGL acts as a potent suppressor of estrogens resulting in severe mucosal atrophy. On the contrary, TAM seems nearly always to display some agonistic hormonal stimulation.