Breast cancer in men in Cote d'Or (France): epidemiological characteristics, treatments and prognostic factors.

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.

Population-based study of breast cancer survival in Cote d'Or (France): prognostic factors and relative survival.

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.

What is the best way to assess microsatellite instability status in colorectal cancer? Study on a population base of 462 colorectal 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.

In situ hybridization detection of single-copy human papillomavirus on isolated cells, using a catalyzed signal amplification system: GenPoint.

The performance and drawbacks of GenPoint, 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 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 appeared the most sensitive method of revelation, easily allowing detection of 1-2 copies of HPV DNA on isolated cells by in situ hybridization.

Factor analysis of confocal image sequences of human papillomavirus DNA revealed with fast red in cervical tissue sections stained with TOTO-iodide.

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.

Four-dimensional factor analysis of confocal image sequences (4D-FAMIS) to detect and characterize low copy numbers of human papillomavirus DNA by FISH in HeLa and SiHa cells.

Visualization 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.

Morphological analysis of in situ hybridization signals in cervical intraepithelial neoplasia containing human papillomavirus type 16 or 18: relationship with histological grade and DNA content.

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.

Detection of low copy numbers of HPV DNA by fluorescent in situ hybridization combined with confocal microscopy as an alternative to in situ polymerase chain reaction.

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.

Laser scanning confocal microscopy and factor analysis of biomedical image sequences (FAMIS) to detect and characterise HPV DNA sequences by FISH in HeLa cells.

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.

Detection of human papillomavirus DNA in genital lesions by enzymatic in situ hybridization with Fast Red and laser scanning confocal microscopy.

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.

[Quantification of protein p53 expression by image analysis in 58 cases of colon carcinoma. Study of correlations between sex, age, histology, Dukes stage, DNA content and lymph node invasion]. / Quantification de l'expression de la protéine p53 par analyse d'images dans 58 cas de carcinomes coliques. Etude des corrélations avec le sexe, l'âge, l'histologie, le stade de Dukes, le contenu en ADN et l'envahissement ganglionnaire.

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.

Benign breast disease: absence of genetic alterations at several loci implicated in breast cancer malignancy.

Benign breast disease (BBD) is a heterogeneous group of benign breast problems that has been associated with breast cancer risk by several investigators. Genetic alterations have been described in breast carcinomas under the headings of loss of heterozygosity (1p, 3p, 7q, 11p, 17p, 17 and 18q), mutations (p53, c-H-ras-1), and/or gene amplifications (c-myc, int-2/FGF3, and c-erbB-2/neu). In an attempt to determine whether these genetic alterations might also be involved in the development of BBD, we have analyzed such alterations in 50 BBD lesions. The histological types of samples studied were: 37 fibroadenomas; 8 benign phyllode tumors; and 5 fibrocytic diseases. Cellular DNA was extracted from tissues and from corresponding blood leukocytes according to standard techniques, digested with appropriate restriction endonucleases, and analyzed by Southern blot. The following are informative cases found in a total number of patients analyzed for each locus: 13 of 26 for L-myc (1p); 9 of 23 for THRB (3p); 11 of 29 for met (7q); 27 of 50 for c-H-ras-1 (11p); 3 of 13 for TP53 (17p); 14 of 50 for D17S30 (17p); 20 of 33 for D17S4 (17q); and 13 of 33 for D18S5 (18q). No loss of heterozygosity was detected at any of the examined loci. Alternatively, none of the 50 BBD cases displayed an amplification of the three genes tested (c-myc, int-2/FGF3, and c-erbB-2/neu). Our results show that molecular alterations, which are more frequently involved in malignant breast carcinomas, do not occur in BBD lesions. These results indicate that these molecular alterations could constitute late events in the pathogenesis of breast carcinomas.

Evaluation of multidrug resistant phenotype by flow cytometry with monoclonal antibodies and functional tests.

Multidrug resistant (MDR) phenotype is characterized by a defect in drug accumulation caused by overexpression of a transmembrane glycoprotein, the P-glycoprotein (P-gp). MDR phenotype can be characterized either with monoclonal antibodies raised against P-gp or with functional tests, most often based on the incorporation of fluorescent compounds. In the present study, data obtained with the monoclonal antibodies C219, JSB1 and MRK16 are compared to those of functional tests performed by flow cytometry including uptake of daunorubicin (DNR), Rhodamine 123 (Rh 123) or Hoechst 33342. Sensitive and resistant cell lines K562S, K562R, KBA1 and KB31, derived either from a human chronic myeloid leukemia or from a human epithelial carcinoma, were used. In resistant cells, P-gp expression was revealed with either the monoclonal antibodies C219, JSB1 or MRK-16. The most specific results were obtained with MRK-16. With functional tests, no matter which dyes were used, the fluorescence was always stronger in sensitive than in resistant cells. However, with DNR and Hoechst 33342, an incorporation of these dyes was exhibited in resistant cells. This phenomenon was not observed with Rh 123, which makes it possible to distinguish clearly between sensitive and resistant cells and to detect as few as 1% of resistant cells. Because of its high sensitivity, the functional test involving incorporation of Rh 123 was successfully used in acute myeloid leukemia to detect multichemoresistant cells.

Alphafetoprotein-producing gastric adenocarcinoma.

We report a rare gastric tumour characterized morphologically by its hepatoid features and alpha-fetoprotein production and which presented clinically with gastric haemorrhage. Gastric fibroscopy showed a bleeding tumour of the antrum. The microscopic appearance of the tumor showed two different patterns. The most extensive presented hepatoid features. The second pattern showed undifferentiated features. The tumour cells showed immunohistochemical positivity for alphafetoprotein, EMA and p53 protein; 37% were aneuploid with a DNA index of 1.46. The serum level of alphafetoprotein was not measured before the gastrectomy but after ten days it was elevated at 1070 ng/ml. The patient died 6 months after the admission. This case provides, for the first time, information on the DNA content and the p53 expression of this unusual and aggressive variant of gastric adenocarcinoma.

Flow cytometry evaluation of the multidrug-resistant phenotype with functional tests involving uptake of daunorubicin, Hoechst 33342, or rhodamine 123: a comparative study.

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.

Multidrug resistance phenotype evaluation by immunofluorescence and functional tests: comparison of two monoclonal antibodies and three fluorescent dyes in three cells lines.

Various agents have been shown to enhance drug sensitivity of multidrug resistant (MDR) cells and are thus of interest when the MDR phenotype is identified. Detection of MDR cells is of importance and can be carried out either by immunofluorescence with monoclonal antibodies or by functional tests using fluorescent dyes uptake. MDR has been analysed by flow cytometry on three sensitive and resistant cell lines, with MRK16 and C219 monoclonal antibodies directed against P-glycoprotein (P-gp) and with rhodamine 123, Hoechst 33342 and daunorubicin. Resistant cells were revealed by MRK16 and C219 but the results obtained with MRK16 gave higher both percentages of fluorescent cells and mean fluorescence. Fluorescence intensity observed with daunorubicin was lower than with rhodamine 123. With Hoechst 33342, mean fluorescence was quite identical on sensitive and on resistant cells. It was concluded that MRK16 and rhodamine 123 were well adapted to detect P-gp and evaluate its functional ability.

[Cancers of the kidney: multiple drug resistance]. / Les cancers du rein: l'obstacle de la chimiorésistance.

The treatment of kidney cancers raise difficult problems. Usual treatments combining surgery, radiation therapy, chemotherapy and hormonotherapy are poorly effective. The immunotherapy gave only an objective response rate of 25% in metastatic renal cell carcinomas. Among cellular resistance mechanisms of renal carcinoma, the multi-drug resistance (MDR) phenotype and the glutathione redox cycle have been studied. The MDR is related to overexpression of a 170 kDa membrane glycoprotein, the so-called P glycoprotein (Pgp). This protein is a pump able to extrude from cytoplasm drugs with various structures and mechanisms. The Pgp is encoded by the MDR1 gene expression is very low in most of the normal tissues, except in colon, liver and kidney. In these tissues, the Pgp would ensure a detoxifying function related to cellular toxic compounds, elimination or transfer of molecules synthesized by the cells. According to the intrinsic resistance of renal carcinoma, MDR1 gene expression has been determined. Approximately, 80% of fresh kidney cancer before chemotherapy express resistance phenotype. Reversal compounds specifically inhibiting Pgp were found, such as verapamil, cyclosporin or quinidine. Unfortunately, the two first compounds give cardiac toxicity and immunosuppression, respectively. So far, clinical trials have not been demonstrating, but no biological resistance measures were determined, in parallel. According to the multifactorial resistance the association of reversals to chemotherapy should be introduced in clinical trials, in correlating clinical response to biological mechanisms of resistance.

Characterization of seven kidney tumors by flow cytometry: analysis of cell cycle, DNA content and P-glycoprotein expression.

Seven kidney tumors obtained from patients aged from 5 to 76 years were analyzed by flow cytometry for cell cycle, DNA content and P-glycoprotein expression involved in multidrug resistance. The DNA index seems to be an important criterion since all the tumors were aneuploid. In a case of clear cell carcinoma, two aneuploid clones were identified. In 5 cases of kidney tumors a high proportion of cells in proliferation (S + (G2 + M)) was observed; it was comprised between 13 and 33%. As for P-glycoprotein it was detected only in few tumor cells (5-15%) respectively in a case of clear cell carcinoma and in a case of Wilms' tumor.

[Prostatic specific antigen and prostatic pathology. Value of preoperative blood assay. Preliminary study apropos of 709 case reports]. / Antigène spécifique de prostate et pathologie prostatique valeur du dosage sérique pré-opératoire. Etude préliminaire à propos de 709 observations.

Prostate specific antigen (PSA) is a prostate tissue marker detected by immunostaining in 97% of specimens examined. Tissue staining is variable and cancers are more heterogeneous than normal or hyperplastic prostate. Serum PSA levels in patients with normal or hyperplastic lesions are 12 ng/ml + 19 and are positively correlated with the weight of the gland. In patients with carcinoma serum PSA levels are 216 ng/ml + 782 and are positively correlated with tumor spread. PSA assay is of little value for screening for prostatic carcinoma. However, carcinoma of prostate is more frequent when PSA levels are above 10 ng/ml and the level of 50 ng/ml indicates capsular penetration, seminal vesicle or lymph node involvement or metastatic spread.