Alain Niveleau

High Epstein-Barr virus serum load and elevated titers of anti-ZEBRA antibodies in patients with EBV-harboring tumor cells of Hodgkin's disease

Hodgkins disease is commonly associated with EBV latent infection. The incidence of EBV reactivation (active infection or EBV infection with replicative cycle) was evaluated in a series of 30 patients with untreated Hodgkins disease (except for one case with chronic lymphocytic leukemia) by quantitation of EBV DNA and titration of anti‐ZEBRA antibodies in serum samples. DNA was detected in serum (>2.5 × 102 genomes/ml) in 15 of 30 patients and was more frequent in Hodgkins disease with EBV‐positive Reed‐Sternberg cells (10/12) than in EBV‐negative cases (5/18), (P < 0.01). Of interest was the demonstration that viremia correlated well with increased titers of anti‐ZEBRA IgG and/or standard serological profiles of EBV reactivation (12/15), (P < 0.05). However the lack of EBV replicative cycle in Reed‐Sternberg cells (negative for ZEBRA antigen and early antigen BHLF1) suggests that the viral replication occurs in a nonneoplastic cell compartment rather than in tumor cells. The measurement of EBV DNA loads and the titration of anti‐ZEBRA antibodies shed new lights on the link between activation of EBV replication and Hodgkins disease: these serological markers together with the determination of the EBV status of the tumor suggest that replication of the viral genome occurs with a decreased efficiency of the immune system, thus allowing progression of the tumor. J. Med. Virol. 57:383–389, 1999.© 1999 Wiley‐Liss, Inc.

Evaluation of global DNA hypomethylation in human colon cancer tissues by immunohistochemistry and image analysis

BACKGROUND Global hypomethylation of DNA is frequently observed in human tumours. This alteration is detected in early adenomas in colorectal tumorigenesis. Information is currently acquired after extraction of DNA from tissues, digestion with nucleases, and analysis by reverse phase chromatography, or treatment with restriction enzymes followed by gel electrophoresis analysis and Southern hybridisation with radiolabelled probes. AIMS The purpose of our work was to evaluate the global methylation status of DNA in malignant lesions without loosing the histopathological features of the samples. PATIENTS The investigation was performed on paired normal-tumour tissues from 13 patients undergoing surgical resection of colorectal adenocarcinomas. METHODS Antibodies raised against 5-methylcytidine can be used to label methyl rich regions in interphase nuclei. This technique was adapted to the study of paraffin embedded tissues and an immunohistochemical method was developed to assess the global methylation status of individual nuclei while preserving cell morphology and tissue architecture. Computer assisted quantification of the staining intensity was performed on malignant and normal zones of human colon tissues to test the correlation between the immunolabelling signal and the respective histological patterns observed. RESULTS Qualitative and quantitative differences were observed and measured between the normal and malignant part of each sample. Morphologically altered nuclei displayed densely labelled spots within faintly labelled areas whereas normal nuclei were darker and uniformly stained. Image analysis allowed calculation of the average integrated optical density of the nuclei in both types of tissues, demonstrating a constant and significantly lower intensity for the former type of cells.

Reduced levels of poly(ADP-ribosyl)ation result in chromatin compaction and hypermethylation as shown by cell-by-cell computer-assisted quantitative analysis

The unmethylated status of the CpG islands is important for gene expression of correlated housekeeping genes since it is well known that their methylation inhibits transcription process. An interesting question that has been discussed but not solved is how the CpG islands maintain their characteristic unmethylated status even though they are rich in CpG dinucleotides. Our previous in vitro and in vivo research has shown that poly(ADP‐ribosyl)ation is involved in protecting CpG dinucleotides from full methylation in genomic DNA and that a block of poly(ADP‐ribosyl)ation is also involved in modifying the methylation pattern in the promoter region of Htf9 housekeeping gene. In this study we locked for cytological evidence that in the absence of an active poly(ADP‐ribosyl)ation the DNA methylation pattern in L929 and NIH/3T3 mouse fibroblast cell lines is altered. For this purpose, differences in the methylation levels of interphase nuclei from control and treated cultures of two murine cell lines preincubated with 2 mM 3‐aminobenzamide, an inhibitor of poly(ADP‐ribosyl)ation, were measured in individual cells after indirect immunolabeling with anti‐5MeC antibodies. The quantitative analysis allowed us to demonstrate that blocking of the poly(ADP‐ribosyl)ation results in a higher number, size, and density of antibody binding regions in treated cells when compared to the controls. Analogously, sequential Giemsa staining and indirect immunolabeling of the same slides showed the hetero‐chromatic regions colocalized with the extended methyl‐rich domains.—de Capoa, A., Febbo, F. R., Giovannelli, F., Niveleau, A., Zardo, G., Marenzi, S., Caiafa, P. Reduced levels of poly(ADP‐ribosyl)ation result in chromatin compaction and hypermethylation as shown by cell‐by‐cell computer‐assisted quantitative analysis. FASEB J. 13, 89–93 (1999)

Quantitation by image analysis of global DNA methylation in human spermatozoa and its prognostic value in in vitro fertilization: a preliminary study

OBJECTIVE To determine the relationship between sperm DNA methylation level and sperm characteristics and pregnancy rates. DESIGN Prospective study. Quantitation by image analysis of DNA methylation in sperm nucleus. SETTING Department of Reproduction Biology, Edouard Herriot Hospital, Lyon, France. PATIENT(S) Infertile couples undergoing IVF-ET. INTERVENTION(S) The immunostaining of 5 methyl-cytosine was performed on the spare sperm suspension that was used for an assisted reproduction technology procedure. MAIN OUTCOME MEASURE(S) Sperm characteristics according to World Health Organization criteria, sperm motility parameters with computer-assisted semen analysis, sperm DNA methylation level, and heterogeneity index (HI). RESULT(S) Sperm DNA methylation level and HI are correlated with sperm DNA characteristics. HI is negatively correlated with fertilization rate; sperm DNA methylation level is correlated with pregnancy rate. CONCLUSION(S) The DNA methylation level in human spermatozoa could be a new approach to evaluating the ability of spermatozoa to fertilize and lead to normal embryo development.

Immunohistochemical Evaluation of Global DNA Methylation: Comparison with in Vitro Radiolabeled Methyl Incorporation Assay

The in vitro radiolabeled methyl incorporation assay, a commonly used technique to evaluate global methylation of DNA, has some disadvantages and limitations. The purpose of the present study was to compare the results of global DNA methylation evaluated by radiolabeled methyl incorporation (CPM/μg of DNA) with immunohistochemical staining of the same tissue sections with a monoclonal antibody developed against 5-methylcytosine (5-mc). We used archival specimens of squamous cell cancer (SCC) of the human lung with a matched uninvolved specimen (n = 18 pairs) and 18 lung specimens from subjects without lung cancer (noncancer specimens) to make this comparison. The immunostaining for 5-mc was reported as a percentage of cells positive for staining as well as a weighted average of the intensity score. The results suggested that both radiolabeled methyl incorporation assay and immunostaining for 5-mc can be used to demonstrate hypomethylation of DNA in SCC tissues compared to matched uninvolved tissues. An advantage of immunostaining, however, is its ability to demonstrate hypomethylation of SCC compared to adjacent bronchial mucosa on the same archival specimen, obviating the need to use sections from both SCC and matched uninvolved tissues. Only by using the immunostaining technique were we able to document a statistically significant difference in DNA methylation between SCC and noncancer tissues. We conclude that the immunostaining technique has advantages over the radiolabeled methyl incorporation assay and may be best suited for evaluation of global DNA methylation when the methylation status of cancer cannot be normalized by methyl incorporation of normal tissues or when the number of samples available for evaluation is small.

Correlation between the DNA global methylation status and progesterone receptor expression in normal endometrium, endometrioid adenocarcinoma and precursors

Endometrial carcinomas are the most common malignancy of the female genital tract and the third most common cancer in women. Progesterone and oestrogen receptors (PRs, ERs) are the most widely documented prognostic and predictive factors in endometrioid adenocarcinoma. Besides the hormonal pathway involved in the progression of preneoplastic and neoplastic lesions, alterations of the DNA methylation status have been shown to be an early signal of tumorigenesis. In this study, we show that in normal endometrium, during the proliferative phase, DNA methylation and PR expression are high, with a significant decline towards the end of the secretory phase and a gradual increase in non-atypical and atypical endometrial hyperplasia; they reach their highest level in grade I, then decrease significantly in grade-II and grade-III endometrioid adenocarcinomas. During each stage, a significant positive correlation is observed between DNA methylation and PR (P<0.0001). The strong parallelism between DNA methylation and PR expression precludes establishing a precise determination regarding the timing of these events, clearly involved in the genesis of endometrioid adenocarcinoma.

Global DNA methylation evaluation: potential complementary marker in differential diagnosis of thyroid neoplasia

The implications of global DNA hypomethylation were recently reported in several models of tumorigenesis. Little is known about this epigenetic event in thyroid neoplasia. The study aimed to evaluate the status of global DNA methylation in several types of thyroid tumors using a monoclonal antibody specific for 5-methylcytidine (5-mc) and to define the diagnosis potential of this marker. 5-mc immunostaining scores were calculated in 17 papillary thyroid carcinomas (PTC), 6 follicular thyroid carcinomas (FTC), 16 follicular adenomas (FA), 19 nodular goiters (NG) and ten Hürthle cells adenomas (HCA). The expression of galectin-3 was also evaluated. Computerized image analysis showed a significant lower level of 5-mc immunostaining in thyroid carcinoma when compared with benign tumors or adjacent normal thyroid parenchyma (P<0.0001). Overall, 5-mc accuracy to distinguish malign from benign thyroid tumors was similar to that of galectin-3 (89% versus 87%, P>0.05). The combination of 5-mc with galectin-3 led to an excellent accuracy level of 96%. Among follicular neoplasia 5-mc accuracy to differentiate malign tumors trends to be higher than galectin-3 one (90% versus 66%, P=0.06). These data stress the necessity of epigenetic events evaluation among thyroid nodules and propose global DNA methylation assessment as a potential diagnostic tool to combine with other valuable markers.

Pattern of nonspecific (or global) DNA methylation in oral carcinogenesis.

Although alterations in nonspecific (or global) DNA methylation (GDM) in specific cells are known to be involved in the process of lung carcinogenesis, similar associations have not been evaluated in other smoking‐related cancers of the head and neck.

Patterns of Global DNA and Histone Methylation Appear to be Similar in Normal, Dysplastic and Neoplastic Oral Epithelium of Humans

Although there is growing interest in the possibility that alterations in histone methylation may play a role in carcinogenesis, it has not been explored adequately in humans. Similarly, there are no reports of associations between this and a similar epigenetic event, DNA methylation. Using immunohistochemical staining, we compared the methylation of DNA and histones in histopathologically normal oral epithelium, dysplastic oral lesions, and squamous cell cancers (SCCs) from subjects with squamous cell cancer (n = 48) with those of normal oral epithelium from subjects without oral cancer (n = 93) who were matched on age and race. Monoclonal antibodies specific for 5 methyl cytosine (5-mc), lysine 4 of histone H3 (H3-Lys4), and lysine 9 of histone H3 (H3-Lys9) were used in this study. The percentages of cells positive and a weighted average of the immunostaining intensity scores were calculated for each of these tissues, and Spearman correlation analyses were employed to study associations between DNA and histone methylation. Correlations between DNA and histone methylation, H3-Lys4 and H3-Lys9 were positive and statistically significant in all tissue types; they were strongest in normal oral epithelium from non-cancer subjects (n = 0.63, p < 0.001 and r = 0.62, p < 0.001 respectively). Similarly, the positive correlations between H3-Lys4 and H3-Lys9 were statistically significant in all tissue types and strongest in normal oral epithelium from non-cancer subjects (r = 0.77, p < 0.001). Patterns of DNA and histone methylation are similar in tissues across the spectrum of oral carcinogenesis, and there is a significant positive association between these two epigenetic mechanisms.

Mandatory fortification with folic acid in the United States is not associated with changes in the degree or the pattern of global DNA methylation in cells involved in cervical carcinogenesis

The purpose of this study was to evaluate whether mandatory fortification of grain products with folic acid in the USA is associated with changes in global DNA methylation in cells involved in cervical carcinogenesis. Archived specimens of cervical intraepithelial neoplasia (CIN) diagnosed before (1990-92) and after mandatory folic acid fortification (2000-02) were used to examine for global DNA methylation in specific lesions involved in cervical carcinogenesis by using a monoclonal antibody specific for 5 methyl cytosine (5-mc). The total number of lesions examined was 152 in the pre-fortification period and 172 in the post-fortification period. Immunohistochemical staining for 5-mc, the assessment of methylation status and data entry were blinded with regard to the fortification status. Age- and race-adjusted mean percentage of cells positive for 5-mc or the 5-mc score was not significantly different (P>0.05) between the pre- and post fortification periods in any of the individual lesions evaluated (i.e., normal cervical epithelium, reactive cervical epithelium, metaplastic cervical epithelium, CIN or carcinoma in situ). The degree of global DNA methylation was significantly higher (P<0.0001) in >or= CIN 2 lesions compared to <or= CIN 1 lesions, regardless of the fortification group. These results suggest that mandatory fortification with folic acid in the United States has not resulted in a change in the degree or the pattern of global DNA methylation in cells involved in cervical carcinogenesis.