La plupart des données sur l'efficacité de l'hormonothérapie proviennent d'essais cliniques randomisés. Peu ou moins de données existent sur l'efficacité de l'hormonothérapie en vie réelle et sa prescription au cours du temps. Cette étude avait pour objectif d'identifier les facteurs pronostiques de la survie chez les femmes atteintes d'un cancer du sein non métastatique à récepteurs hormonaux positifs (RH +). Une étude observationnelle rétrospective a été réalisée à partir du Registre des cancers du sein et autres cancers gynécologiques de Côte d'Or en France. Toutes les femmes ayant reçu un diagnostic de cancer du sein RH + invasif primaire non métastatique entre 1998 et 2015 et traitées par hormonothérapie ont été incluses. Les patientes dont le statut RH ou le type d'hormonothérapie était inconnu ont été exclues. L'hormonothérapie administrée dans le cadre d'une métastase ou d'une rechute n'a pas été prise en compte. Les femmes atteintes de tumeurs bilatérales ont également été exclues. La survie sans maladie a été estimée à l'aide de la méthode de survie ajustée directe. Les facteurs pronostiques de la survie ont été identifiés à l'aide d'un modèle de Cox. Une régression logistique multivariée a été également effectuée pour déterminer les facteurs associés au choix du type d'hormonothérapie. Environ 3976 femmes traitées par hormonothérapie pour un cancer du sein non métastatique RH + ont été incluses. L'âge, le type d'hormonothérapie, le grade SBR, le traitement, le stade et les comorbidités étaient indépendamment associés à la survie sans maladie. Les femmes n'ayant reçu ni chirurgie ni radiothérapie avaient le plus grand risque de récidive (HR = 3,78, IC à 95 % [2,83–5,06], p < 0,0001). Les femmes traitées par un inhibiteur de l'aromatase (IA) avait un risque réduit de récidive (HR = 0,80, IC à 95 % [0,67–0,95], p = 0,01) comparées à celles traitées par tamoxifène. Comparées aux femmes avec un index de comorbidité de Charlson (CCI) égal à 0, les femmes avec un CCI ≥ 1 étaient plus susceptibles de recevoir un IA (OR = 1,74, IC à 95 % [1,31-2,31], p = 0,0001). Le type histologique et le type de chirurgie étaient également des prédicteurs du choix du type d'hormonothérapie. En effet, les femmes atteintes d'un cancer lobulaire (OR = 1,37, IC à 95 % [1,05-1,78], p = 0,0204) étaient plus susceptibles de recevoir un IA comparativement à celles atteintes d'un cancer canalaire. Les comorbidités, l'âge au moment du diagnostic et le fait de recevoir un traitement par chirurgie en association à la radiothérapie étaient des prédicteurs de la survie chez les femmes atteintes d'un cancer du sein traitées par hormonothérapie. Cette étude montre également que les femmes ayant reçu du tamoxifène pour leur cancer avaient plus de risque de récidives comparées aux femmes traitées par IA. Les comorbidités, le type histologique et le type de chirurgie étaient des prédicteurs du choix de la classe d'hormonothérapie.

The purpose of this study was to examine the implications of childhood community contexts in the U.S. for physical health problems related to impaired metabolic conditions, and coronary/cardiovascular diseases during young adulthood. Data came from Waves 1 and 4 (1995 and 2008) of the National Longitudinal Study of Adolescent Health (N = 11,845). Multilevel logistic-normal regression was used to examine the relative risk or odds ratios of physical health problems in young adulthood (2008), based on both 1990 census level and 1995 survey data. Childhood community disadvantage and minority concentration increased the risk of young adult obesity, hypertension, diabetes, and high cholesterol. However, the influence of both community disadvantage and minority concentration on young adult physical health outcomes differed by race/ethnicity. Our findings clearly point to the increased risk of physical health problems related to coronary and cardiovascular diseases when a child is raised in an adverse and minority concentrated community. This influence of the community was pervasive and independent of family characteristics. Programs should combat adverse community conditions and enhance resiliencies of youth and families living in such communities.

Breast cancer in men is rare, and clinical trials are thus not feasible. This study aimed to describe the epidemiological characteristics, treatment and prognostic factors of breast cancer in men. A population-based study was performed using data from the Cote d'Or breast and gynaecological cancer registry. Data on male breast cancer diagnosed from 1982 to 2008 were provided. Relative survival rates were estimated at 5 years according to the characteristics of the patient and tumour, and treatment. Prognostic factors of survival in men with breast cancer were identified using a generalised linear model. Seventy-five men with invasive breast cancer were registered. Mean age at diagnosis was 66 years. The use of adjuvant chemotherapy (P= 0.013) and hormone therapy (P < 0.0001) increased over time. Relative survival rate at 5 years was 69% for the whole population. Analysis of relative survival according to the treatment showed that survival was longer for patients treated with surgery + radiotherapy + hormone therapy: 89% at 5 years. Scarff, Bloom and Richardson grade was independent prognostic factor of survival. Male breast cancer is a rare disease with a poor prognosis, and diagnosis is often made at an advanced stage. Early diagnosis and better knowledge of the disease would certainly lead to improvements in the prognosis.

Les cancers coliques présentant le phénotype CIMP (CpG island methylator phenotype) possèdent des caractéristiques anatomocliniques et moléculaires spécifiques et proches de celles des cancers MSI-High (MicroSatellite Instability High). La plupart des études portant sur la méthylation ne distinguent que 2 groupes de cancers : hautement méthylés (CIMP-High) et peu ou pas méthylés (CIMP-Low ou No-CIMP). Dans notre étude, nous avons défini trois sous groupes de méthylation (No-CIMP, CIMP-Low et CIMP-High) et évalué leur valeur pronostique sur une base de population de cancers coliques sporadiques. Un total de 582 adénocarcinomes coliques a été caractérisé par MSP fluorescente (methylation specific PCR) à l'aide de 5 marqueurs (hMLH1, P16, MINT1, MINT2, MINT31). Le statut No-CIMP a été défini par l'absence de méthylation des marqueurs étudiés, le statut CIMP-Low lorsque 1 à 3 marqueurs étaient méthylés, et CIMP-High lorsque 4 à 5 marqueurs étaient méthylés. Le statut de méthylation a été confronté aux données anatomocliniques et moléculaires (statut MSI, mutation de BRAF et KRAS). La survie globale et relative des patients a été évaluée en fonction du statut de méthylation. Dans le groupe MSS (MicroSatellite Stable), le statut CIMP-High a été retrouvé significativement associé à une localisation proximale (p = 0,011), à la mutation de BRAF (p < 0,001). Les mutations de KRAS étaient plus associées aux statuts CIMP-High et CIMP-Low (p = 0,008). A 5 ans la survie relative des patients porteurs d'une tumeur MSS était d'autant plus faible que leur tumeur était méthylée (64 % pour les No-CIMP, contre 50,6 % pour CIMP-Low et 37,7 % pour CIMP-High). Ces résultats restaient significatifs en analyse multivariée ajustée sur l'âge, le stade, et le statut mutationnel de BRAF et KRAS (CIMP-Low : Hazard-Ratio = 1,85 ; IC 95 % : [1,37 - 2,51] ; CIMP-High : Hazard-Ratio = 2,90 ; IC 95 % : [1,53 - 5,49] comparé à No-CIMP). Chez les patients porteurs d'une tumeur MSI-H, aucune différence de survie n'a été observée en fonction de la méthylation. Notre travail démontre que les patients des groupes No-CIMP et CIMP-Low et porteurs d'une tumeur MSS ont des caractéristiques anatomocliniques similaires mais une survie différente. Cette étude souligne l'effet pronostique péjoratif de la méthylation chez les patients MSS et l'importance majeure d'identifier les 3 groupes de méthylation.

Des micrométastases ou cellules isolées tumorales peuvent être détectées en analyse anatomopathologique avec éventuellement l'aide de coloration par immuno-histochimie au niveau des ganglions, de la moelle osseuse et du sang. Ces atteintes de toute petite taille ont-elles une réalité clinique, c'est-à-dire un impact pronostique et en conséquence un impact sur les décisions thérapeutiques ? L'impact de la détection de micrométastase des ganglions sentinelles est discuté avec deux éléments : 1) le taux de ganglions non sentinelles (GNS) envahis au curage axillaire complémentaire est-il différent de celui des GS pN0 et de celui des GS pN1a ? 2) les survies sont-elles significativement différentes en fonction de cette atteinte des GS ? Le risque d'atteinte des GNS en cas de micrométastase ou de cellules isolées est différent de celui des cas où les GS sont indemnes (risque de 7 à 8 %) et de celui des cas où les GS sont envahis par une macrométastase (risque de 30 à 50 %). La présence de micrométastase a un impact pour les cancers du sein sur le risque d'envahissement d'autres ganglions du creux axillaire, possiblement sur la survie globale ou sans récidive ainsi que sur l'indication de traitement adjuvant de chimiothérapie et/ou de radiothérapie (sur les champs de traitement des aires ganglionnaires régionales). Il s'agit donc bien d'une réalité clinique, même si la question du bénéfice du curage axillaire complémentaire reste posée.Micrometastases or sub-micrometastases can be detected by standard histopathological method sometimes associated with immunohistochemistry in lymph nodes, bone marrow and blood. The consequence of these small size involvement may be prognostic and therapeutic. Two factors are necessary to assess this kind of involvement: the rate of involvement of non-sentinel lymph node after axillary lymph node dissection and significative difference of survivals. The rate of involvement of non-sentinel lymph node in case of micrometastases or sub-micrometastases is different from the rate of involvement in case of no lymph node metastases (7 to 8%) or in case of macrometases (30 to 50%). Micrometastase is an important factor to determine the rate of involvement of non-sentinel lymph node, the overall or disease free survival and to assess the need of radiotherapy and chemotherapy. In conclusion, micrometastases and sub-micrometastases have a clinical impact even if complementary axillary lymph node dissection is still discussed.

BACKGROUND:Few population-based studies have reported jointly analyses of relative survival according to the following prognostic factors: tumour-node-metastasis (TNM) stage, age, number of examined and positive nodes, hormonal status, histological Scarff, Bloom and Richardson (SBR) grade, tumour extension, hormone receptor status and tumour multifocal status.PATIENTS AND METHODS:Data on female invasive breast cancer were provided by the Cote d'Or breast cancer registry. The Kaplan-Meier method and log-rank test were used to estimate and compare the survival probability at 1, 5, 10 and 15 years. The effect of prognostic factors on survival was assessed with crude and relative multivariate survival analyses.RESULTS:Crude survival seemed to be worse in patients aged >60 years compared with those aged 45-60 (P > 0.0001), whereas relative survival did not differ. TNM stage, histological SBR grade, progesterone receptor status, tumour multifocal status, locoregional extension and the period of diagnosis were independent prognostic factors of crude and relative survival.CONCLUSION:Breast cancer is influenced by many factors. Despite the absence of any association between the number of examined nodes and overall survival in this study, the number of nodes removed, in conjunction with other prognostic factors, may be useful in selecting node-negative patients for systemic therapy.

PURPOSE Prognostic factors that could select high-risk recurrence colorectal cancer patients and predict chemosensitivity are needed. Since mutations of mitochondrial DNA (mtDNA) have been described in different types of cancers and since they may play a role in response to anticancer agents, we investigated in a population-based series of colorectal cancer patients the clinical value of mtDNA mutations. PATIENTS AND METHODS The displacement loop (D-loop) region of mtDNA was sequenced on a series of 365 patients recorded in the Digestive Cancer Registry of Côte-d'Or (France) between 1998 and 2000. Clinicopathologic characteristics were correlated to the presence of a D-loop mutation. Survival rates were compared with the log-rank test. A multivariate survival analysis was performed. RESULTS D-loop mutations were found in 38.3% of the tumors. The 3-year survival rate was 53.5% in patients with D-loop mutation versus 62.1% in patients without (P = .05). After adjustment for age, stage, and microsatellite instability status, the relative risk of death in patients with D-loop mutation was 1.40 (95% CI, 1.02 to 1.93; P = .034) as compared with those without. In stage III colon cancers, adjuvant chemotherapy was beneficial only for patients without D-loop mutation (3-year survival, 78.3% v 45.4%, P < .02). In those with D-loop mutation who received adjuvant chemotherapy, the relative risk of death was 4.30 (95% CI, 1.23 to 15.00; P < .02). CONCLUSION The D-loop region is a hotspot for somatic mutations in colorectal tumors. Moreover, presence of tumor D-loop mutation appears to be a factor of poor prognosis in colorectal patients and a factor of resistance to fluorouracil-based adjuvant chemotherapy in stage III colon cancers.

The assessment of the microsatellite instability (MSI) status in colorectal cancers is presently warranted for three reasons: 1) as a screening tool for hereditary nonpolyposis colorectal cancer, 2) as a prognostic marker, and 3) as a potential predictive factor of chemotherapy response. The aim of this study was to evaluate, on a large scale with tissue samples coming from a number of different sources, the difficulties met with routine use of immunohistochemistry (IHC) and to determine if it really does offer an accurate alternative to PCR genotyping. Colorectal carcinomas from 462 consecutive patients resected in public or private hospitals were assessed for MSI status by two methods: MSI testing (with BAT-26 microsatellite) and IHC detection of hMLH1, hMSH2, and hMSH6 proteins. Of the 398 cancers tested, immunohistochemistry was noncontributory in 42 (10.5%), focal in 9 (2.3%), and discordant with the PCR results in 36 (9%). For these 87 cases, complementary analyses were performed to explain discrepancy. After additional IHC assay with modified processing protocols, 8 cases remained noncontributory, 2 focal, and 28 discordant: 18 microsatellite stability IHC/MSI PCR and 10 MSI IHC/microsatellite stability PCR. For these discordant cases, we performed a multiplex PCR assay on DNA extracted from the frozen sample and BAT-26 was amplified from DNA extracted from the paraffin blocks used for IHC. Four discordant cases were reclassified after PCR multiplex assay (3 as MSI and 1 as microsatellite stability). Five other cases displayed intratumoral heterogeneity and 19 remained discordant. The discrepancy could be partly explained by variable technical protocols of fixation in the different laboratories, leading to variations in staining quality and difficulties in IHC interpretation. This population-based study is the first one to show that IHC is not sensitive and specific enough to be used routinely. Immunohistochemistry analysis of MMR proteins must be performed in standardized conditions and interpreted by confirmed pathologists. It cannot replace PCR as long as protocols are not optimized and harmonized.

There are few population based data about lymphadenectomy practices in resections for stomach carcinoma. The aim of the current study was to describe these practices and to determine how many nodes must be examined in order to accurately stage gastric carcinoma.

The performance and drawbacks of GenPoint(TM), which is a catalyzed signal amplification system for immunohistochemistry, have been evaluated for its ability to reveal human papillomavirus (HPV) DNA detected by in situ hybridization with biotinylated DNA probes. For this aim, formalin-fixed cell deposits from carcinoma cells of the uterine cervix, CaSki, SiHa, and HeLa, containing, respectively, 600 copies of HPV DNA type 16, 1-2 copies of HPV DNA type 16, and 10-50 copies of HPV DNA type 18, were used, and the GenPoint(TM) method (consisting of successive incubations with peroxidase-conjugated streptavidin, biotinyl tyramide, and peroxidase-conjugated streptavidin) was compared to immunoenzymatic revelation procedures involving either a one-step reaction (streptavidin-alkaline phosphatase or streptavidin-peroxidase), or a three-step reaction (anti-biotin mouse monoclonal antibody, rabbit anti-mouse antiserum, and mouse APAAP complex). In these conditions, after analysis with a bright-field microscope. GenPoint(TM) appeared the most sensitive method of revelation, easily allowing detection of 1-2 copies of HPV DNA on isolated cells by in situ hybridization. (C) 2001 Wiley-Liss, Inc.

OBJECTIVE:To visualize and localize specific DNA sequences by fluorescence in situ hybridization, confocal laser scanning microscopy (CLSM) and factor analysis of biomedical image sequences (FAMIS).STUDY DESIGN:Human papillomavirus (HPV) DNA was identified in cervical tissue sections with biotinylated DNA probes recognizing the whole genome of HPV DNA types 18 and 16, and DNA-DNA hybrids were revealed by streptavidin-alkaline phosphatase and Fast Red (FR). Cell nuclei were counterstained with TOTO-iodide. Image sequences were obtained using successive dynamic or spectral sequences of images on different optical slices from CLSM. The location of fluorescent signals inside tissue preparations was determined by FAMIS and/or selection of filters at emission. Image sequences were summarized into a reduced number of images, called "factor images," and curves, called "factors." Factors estimate spectral patterns and depth emission profiles. Factor images correspond to spatial distributions of the different factors.RESULTS:We distinguished between FR and nucleus staining in HPV DNA hybridization signals by taking into account differences in their spectral patterns and improved visualization by taking into account differences in their focus (depth emission profiles).CONCLUSION:FAMIS, together with CLSM, made possible the detection and characterization of HPV DNA sequences in cells of cervical tissue sections.

SummaryVisualization and localization of specific DNA sequences were performed by fluorescence in situ hybridization, confocal laser scanning microscopy (CLSM), and four‐dimensional factor analysis of biomedical image sequences (4D‐FAMIS). HeLa and SiHa cells containing, respectively 20–50 and 1–2 copies per cell of human papillomavirus (HPV) DNA type 18 and 16 integrated in cellular DNA were used as models. HPV‐DNA was identified using DNA probes containing the whole genome of HPV‐DNA type 18 or 16, and DNA–DNA hybrids were revealed by alkaline phosphatase and Fast Red. Cell nuclei were counterstained with thiazole orange (TO) or TOTO‐iodide. 4D image sequences were obtained using successive dynamic or spectral sequences of images on different optical sections from CLSM. The location of fluorescent signals within the preparations was determined by FAMIS. This original method summarizes image sequences into a reduced number of images called factor images, and curves called factors. Factors estimate different individual physical behaviours in the sequence such as extinction velocity, spectral patterns and depth emission profiles. Factor images correspond to spatial distributions of the different factors. We distinguished between Fast Red and nucleus stainings in HPV‐DNA hybridization signals by taking into account differences in their extinction velocities (fluorescence decay rate) or spectral patterns, and in their focus (depth emission profiles). In HeLa cells, factor images showed that Fast‐Red‐stained targets could be distinguished from nucleus stainings, and were located on different focal planes of the nuclei. In SiHa cells, 4D‐FAMIS determined as few as 1–2 copies per cell of HPV‐DNA type 16 located in continuous focal planes. Therefore, 4D‐FAMIS, together with CLSM, made the detection and characterization of low copy numbers of genes in whole cells possible.

Taken together TS and p53 have been reported to be predictive of survival and the response to 5FU-based therapy of patients (pts) with colorectal carcinoma.To determine the relationship between TS and p53 expression and their biological significance in CC we evaluated the expression of TS in 143 and p53 in 166 archival paraffin embedded samples of 260 B2 and C Dukes colon cancer pts who were included in a French adjuvant trial (FFCD 8802).In this trial 5FU/Folinic Acid adjuvant chemotherapy (CT) was compared with surgery alone and the results published in a prospective pooled analysis (IMPACT.Lancet 1995, 345 : 939-44) showed the efficacy of 5FU-FA adjuvant Ct (3-yr survival).TS and p53 were stained with the monoclonal antibodies TS-106 (Johnston.Cancer Res 1992, 52 : 4306-12) and DO7 respectively.TS was assessed in terms of low versus high levels of expression and found to be present in high levels in view of the intense positive cytoplasmic staining observed in >-30% of the tumoral cells.Based on the p53 staining observed in >-20% of the nuclei, the tumors were classified as p53 overexpression.The male/female ratio was 138/122 and the Duke's B2/C ratio was 169/91.With a median follow-up time of 83 months, a trend towards an improvement in the EFS was observed in pts receiving adjuvant CT (7-yr EFS: 68.1 vs 59.9%; p=0.069).The differences were statistically significant in Duke's C pts (61.6 vs 32.9%; p=0.027) but not in B2 pts (71.3 vs 71.6%; NS).The prognostic factors (Cox model) were parietal invasion (T4/T3: OR:3.13; p= 0.0008), nodal involvement (1-3 nodes/0:OR:1.71; p=0.024 ->_4 nodes/0: OR:3.17 ; p= 0.003), tumor site (left/right colon: OR:2.48; p= 0.0002) and sex (M/F: OR:1,83; p = 0.0044).Chemotherapy was borderline significant (OR: 0.71 ; p= 0.10) and TS and p53 expression were not significant.Upon examining the risk of event before 2 years, the independent prognostic factors (logistic regression) were found to be nodal involvement (>-_4/0-3 nodes: OR: 8.34 ; p= 0.0038), TS expression (high/low: OR:4.51 ; p= 0.0137), p53 overexpression (yes/no OR:3.84 ; p= 0.0113) and macroscopic appearance (infiltrating/protuberant: OR:3.14 ; p= 0.055).CT was not correlated with this risk.In this study, TS and p53 were found to be significant independant predictors in early risk of event for CC, but their validity decreases with time.The factors do not seem however to be correlated with the outcome of adjuvant CT at the beginning of the treatment; whereas the treatment itself is a favorable long-term prognostic factor in Duke's C patients.

In genital lesions infected by human papillomavirus (HPV), histological criteria and HPV DNA typing are of prognostic value. Therefore, non-radioactive methods such as in situ hybridization are used extensively since they preserve the histological organization of the tissue, and allow the detection and characterization of HPV DNA. However, the sensitivity of these methods is often limited to detection of low copy numbers of HPV DNA in isolated cells or in tissue sections, and therefore alternative techniques have been explored. In the present study, 1–2 copies of HPV DNA were visualized in SiHa cells either by in situ amplification of nucleic acid sequences with the polymerase chain reaction (PCR) or by fluorescent in situ hybridization (FISH) associated with observation by laser scanning confocal microscopy (LSCM). The latter procedure was evaluated for use on histological tissue sections to identify low copy numbers of HPV DNA. Genital lesions which were negative by enzymatic in situ hybridization and FISH but histologically suspected of HPV infection were investigated, and intense signals were obtained both with in situ PCR and with the combined use of FISH and LSCM. Therefore, the combination of FISH with LSCM examination may be as valuable as in situ PCR to detect viral genes present in small amounts in isolated cells and in tissue sections.

Among 345 lesions histologically defined as cervical intraepithelial neoplasia (CIN) examined by in situ hybridization (ISH) for the presence of DNA from human papillomavirus (HPV) types 6/11, 16, 18, 31, 33, and 51, a group of 69 lesions (41 low grade and 28 high grade) containing HPV 16 or 18 was further characterized with the following criteria: DNA ploidy and morphological patterns of ISH spots, i.e., punctate or diffuse throughout the nuclei corresponding to integrated or episomal state of HPV DNA, respectively. The highest percentage of aneuploid lesions, the highest diploid index values, and the highest proportion of CIN with punctate ISH signals were associated with high-grade lesions. In addition, punctate ISH signals were also most frequently found in aneuploid CIN. These results underline that punctate ISH signals considered as integrated HPV DNA were preferentially associated with aneuploid and high-grade lesions, and lead to suggest that this later criteria could be used to predict the evolution of a lesion towards malignancy.

Human papillomavirus (HPV) infection with potentially oncogenic types 16 or 18 is common in genital lesions especially in uterine carcinomas. In such lesions, in situ hybridization with non-radioactive probes is a powerful tool for the histopathologist to detect and type HPV DNA either on cell deposits or on tissue sections. The use of an immunohistochemical method involving alkaline phosphatase and Fast Red TR salt/naphthol AS-MX phosphate is proposed for use with conventional bright-field or fluorescence microscopy as well as by laser scanning confocal microscopy. The alkaline phosphatase-Fast Red reaction has the advantage of producing a red precipitate that permits the detection of in situ hybridization signals by bright-field microscopy, and of obtaining a strong red fluorescence characterized by a lack of bleaching when excited by a green light. Therefore, the alkaline phosphatase-Fast Red reaction is well adapted for observations by fluorescence and confocal microscopy, the latter method allowing the detection, in tissue sections of cervical intraepithelial lesions, of small punctate and large diffuse hybridization signals, considered as integrated and episomal states of HPV DNA respectively. The combination of in situ hybridization with the alkaline phosphatase-Fast Red reaction and confocal microscopy is particularly convincing when hybridization signals are of small size and/or of low fluorescence intensity, especially if they are present in various focal planes; in such conditions, infected cells are easily detected by three-dimensional reconstruction. Therefore, this combination is a suitable method for identifying and characterizing HPV DNA in cells and tissue sections.

The analysis of p53 expression has been performed on 58 cases of colonic carcinomas. p53 expression was revealed by immunohistochemistry with the monoclonal antibody DO.7, and its quantification was performed by image analysis on deparaffinized tissue sections treated by microwaves. p53 expression evaluated by images analysis has been compared to visual estimation performed under light microscopy, and an excellent correlation was found between the two methods. No statistical significant relationships were found between the proportion of tumours expressing p53, sex, age (< 70 or > 70 years), histology (well, intermediate or poorly differentiated) and DNA index (< 1.3 or > 1.3) whereas the proportion of tumors expressing p53 was significantly higher in case of metastatic behaviour as well as in the C stage of Dukes classification. p53 expression was also significantly more important in colonic carcinomas with DNA index higher than 1.3 and in case of metastatic behaviour. According to these data, the metastatic behaviour is correlated both with the proportion of tumors expressing p53 and with the level of p53 expression.

Visualisation and localisation of specific DNA sequences were performed by fluorescence in situ hybridisation (FISH), confocal laser scanning microscopy (CLSM), and factor analysis of biomedical image sequences (FAMIS). HeLa cells containing 10-50 copies per cell of human papillomavirus (HPV) DNA type 18 integrated in cellular DNA were used as a model. HPV-DNA was identified by DNA probes and DNA-DNA hybrids were revealed by alkaline phosphatase and Fast Red (FR) TR salt/naphtol-MX phosphate. Cell nuclei were counterstained with thiazole orange (TO). FAMIS summarises image sequences into a reduced number of images called factor images and curves called factors. Factor images correspond to spatial distributions of the different factors. Factors estimate different individual physical behaviours in the sequence (extinction velocity, spectral emission, depth emission profiles). We verified that HPV-DNA hybridisation signals are specific to the spectrum of FR, and distinguished between FR and TO. The latter result was found by taking into account differences in their extinction velocities. The focus of CLSM was improved on 3D image sequences, and the location of fluorescent signals inside the preparations was determined. Factor images showed that FR stained targets were located on different focal planes at the periphery of the nuclei.

Multidrug-resistant (MDR) cells are characterized by a defect in drug accumulation caused by overexpression of a transmembrane glycoprotein, the P-glycoprotein (P-gp). The MDR phenotype can be characterized either by use of monoclonal antibodies raised against P-gp or with functional tests based on the intracellular accumulation of fluorescent molecules. The aim of the present study was to compare the effectiveness of functional tests performed by flow cytometry including uptake of daunorubicin (DNR) (2 micrograms/ml), Hoechst 33342 (5 micrograms/ml), or rhodamine 123 (RH 123) (0.1 microgram/ml); and to evaluate the effect of cell death induced by heating at 60 degrees C for 2 h on incorporation of DNR and RH 123. Sensitive and resistant human hematopoietic K 562 cells expressing P-gp were identified by monoclonal antibodies C 219 and MRK-16. Fluorescence of the dyes was always higher in sensitive than in resistant cells. However, DNR and Hoechst 33342 produced a slight incorporation in resistant cells, while RH 123 showed lack of incorporation in resistant cells. Thus, RH 123 allows sensitive and resistant cells to be clearly distinguished. In case of cell death, accumulation of RH 123 and DNR were different. With RH 123, fluorescence intensity strongly decreased in sensitive cells. With DNR, fluorescence intensity was enhanced in resistant cells. Thus, when the MDR phenotype is defined by uptake of DNR or RH 123, artifactual results due to cell death may be avoided by using a dye such as propidium iodide to eliminate dead cells.